Category Archives: Stem Cell Treatment

Stem Cell Network Warns that Claims of Stem Cell Treatments for COVID-19 Are Unfounded and Misleading – Benzinga

OTTAWA, March 31, 2020 (GLOBE NEWSWIRE) -- In recent weeks, a number of claims have been made that stem cells can be used as a treatment for the coronavirus disease (COVID-19). Globally, there is no clinically tested and government approved stem cell-based treatment for COVID-19.

The Stem Cell Network (SCN) urges extreme caution to those who are considering purchasing products or services advertised as a preventative or curative treatment for COVID-19. In alignment with other international stem cell and regenerative medicine organizations, SCN strongly opposes the marketing of unproven therapies and urge consumers and patients to consult with their doctor or specialist if they have questions or concerns about their health. The best way to combat the spread COVID-19 is to follow the careful advice given by Canada's Chief Public Health Officer.

Researchers across the globe are collaborating and working hard to find legitimate treatments for COVID-19, but this will take time. It is important to note that when a treatment does become available, it will be announced through recognized medical authorities, such as the World Health Organization, which is coordinating global efforts and actively compiling a database of published research on COVID-19.

For the most up-to-date information on COVID-19, please consult:World Health OrganizationPublic Health Agency of Canada

To learn more about clinical trials or stem cells:Stem Cell Network Clinical Trial FAQsCloser Look at Stem Cells

About the Stem Cell NetworkTomorrow's health is here. The Stem Cell Network (SCN) is a national non-profit that supports stem cell and regenerative medicine research, training the next generation of highly qualified personnel, and delivering outreach activities across Canada. SCN's goal is to advance science from the lab to the clinic for the benefit of Canadians. SCN has been supported by the Government of Canada since inception in 2001. This strategic funding, valued at $118M has benefitted approximately 170 world-class research groups and 3,000 trainees and has catalyzed 23 clinical trials. stemcellnetwork.ca

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Stem Cell Network Warns that Claims of Stem Cell Treatments for COVID-19 Are Unfounded and Misleading - Benzinga

FibroGenesis Files Expanded Patent Coverage for its Fibroblast Cell Therapy to treat Coronavirus (COVID-19) ARDS – Yahoo Finance

Company Reports Potent Synergy Between its Fibroblast-Based Cell Therapy and Hydroxychloroquine

HOUSTON, April 1, 2020 /PRNewswire/ --FibroGenesis, a Texas-based regenerative medicine company focused on tissue regeneration and chronic disease reversal using Human Dermal Fibroblasts (HDFs), today announced the filing of United States Provisional Patent Number 63/002,134 titled, "Peptides and Adjuvants for Augmentation of Fibroblast Therapy for Coronavirus."

FibroGenesis Logo (PRNewsfoto/FibroGenesis)

The claims in the patent include utilization of fibroblast cells along with adjuvants such peptides and hydroxychloroquine which stimulates the production of natural interferon to suppress the viral infection and corresponding "cytokine storm." In one embodiment, the invention provides methods of preventing infection, propagation, and pathology caused by the COVID-19 virus. Also included are claims to modify fibroblasts to express enhanced levels of "therapeutic cytokines."

"As we continue our accelerated preclinical program, we are discovering the superiority of fibroblasts over mesenchymal stem cells and the data is leading us toward multiple treatment options for the patient," commented Tom Ichim, Ph.D., Chief Scientific Officer of FibroGenesis. "By including adjuvants such as peptides and hydroxychloroquine in our treatment we've seen added potency."

"We are working to expand our discoveries in the lab and accelerate the clinical development into a cure for COVID-19 using our advanced fibroblast cell therapy," said Pete O'Heeron, Chief Executive Officer, FibroGenesis. "The war we are fighting, with this invisible enemy, will likely require a cocktail-based approach for victory. At FibroGenesis we are following the lead of Thomas Edison when he discovered the filament for the lightbulb; we are testing as many therapeutic combinations as possible, in search of the most efficient and effective cure. Enhancing the natural production of interferon combined with our previous work can be seen as a possible advancement toward a cure."

About COVID-19 Induced ARDS

Acute respiratory distress syndrome (ARDS) is a type of severe acute lung dysfunction affecting all or most of both lungs and can be a severe complication of viral infections including COVID-19.

It is known that ARDS is often associated with fluid accumulation in the lungs. When this occurs, the elastic air sacs (alveoli) in the lungs fill with fluid and the function of the alveoli is impaired. The result is that less oxygen reaches the bloodstream, depriving organs of the oxygen required for normal function and viability. Severe shortness of breath, the main symptom of ARDS, usually develops within a few hours to a few days after the precipitating injury or infection. There are currently no effective pharmacologic therapies for treatment or prevention of ARDS. Protective lung ventilator strategies remain the mainstay of available treatment options. Due to the significant morbidity and mortality associated with ARDS and the lack of effective treatment options, new therapeutic agents for the treatment of ARDS are needed.

About FibroGenesis

Based in Houston, Texas, FibroGenesis, is a regenerative medicine company developing an innovative solution for chronic disease treatment using human dermal fibroblasts. Currently, FibroGenesis holds 186 U.S. and international issued patents/patents pending across a variety of clinical pathways, including Disc Degeneration, Multiple Sclerosis, Parkinson's, Chronic Traumatic Encephalopathy, Cancer, Diabetes, Liver Failure and Heart Failure. Funded entirely by angel investors, FibroGenesis represents the next generation of medical advancement in cell therapy.

Visit http://www.Fibro-Genesis.com.

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SOURCE FibroGenesis

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FibroGenesis Files Expanded Patent Coverage for its Fibroblast Cell Therapy to treat Coronavirus (COVID-19) ARDS - Yahoo Finance

Cork man shares hacks for isolation that stopped him from ‘going insane’ – Echo Live

I WILL start this by saying Im not an expert by any stretch of the imagination, but having spent 12 days in isolation in October, I wanted to share some of the things that stopped me from going insane!

Try where you can to stick to as regular a routine as possible. I made sure I was out of bed by a certain time each day and always started the day with a loose plan of things I wanted to achieve.

I was in a tiny hospital room so this might sound insane but it really made a difference. Knowing I couldnt leave the room was tough but also knowing there were still elements of my life I could partake in was great and got me out of bed.

This will depend on how well youre feeling but I made sure I did at least 20 minutes of exercise every day. One of the things that can happen when your movement is restricted is that you can start to loose muscle mass so anything from Yoga to star jumps to walking up and down the stairs will help keep you moving and get the blood flowing.

It will also stand to you when you leave isolation as you wont be prepared for how exhausted you will feel doing what was normal to you before you checked into Costa Del Corona.

I know I was coming off the back of intensive Chemotherapy and had no immune system so my symptoms will be different to yours but I remember the mere act of going to Tesco just five minutes from my house resulted in perfuse sweating, light headedness and a two hour nap.

Unless you cant keep anything down, eat even if you dont feel like it. If you are fighting a virus your body will need some fuel.

Try where possible to eat healthy but the main thing is that you eat. I went through three days after Chemo where I had no mind for food but I still eat a little bit of toast a few times a day as I knew it would help.

As much as you need a routine you will also need rest. Listen to your body and let it guide you. Being in a small space unable to leave is actually quite exhausting. If youre also feeling unwell you will be more exhausted then usual so dont feel guilty about a duvet day.

Its important to stay as active as possible but dont give yourself too hard a time if you find yourself napping far more often than usual. This is all part of the recovery.

Although I was in isolation, as long as they were healthy I was allowed visitors. I was also fortunate to have a long line of doctors and nurses coming in and out which made it all the more bearable.

I realise if you are self-isolating this wont be the case but I believe, and this may change, that as long as the person is healthy and they stay one meter away from you, visitors should be allowed and encouraged.

It made such a difference to me to have face to face visitors and phone calls etc as it reminded me that there was a world outside isolation. I did limit the number of visitors per day and some days just said no to anyone popping by but it really did help.

Social media may make you feel connected but nothing beats a voice on the other end of the phone or a smiling face sitting opposite you (one meter away) with a cuppa.

What I also felt really helped was focusing the conversation on the visitor. Giving them the chance to really tell me what was going on in their life especially if things werent great as it made me feel somewhat useful that I could give them an avenue to talk.

This is the biggest battle you will face and where you need to pay the most attention.

The mere fact you cant leave your room / house is enough to send anyone gaga but the fear that will inevitably come with it when you are ill will make it that bit harder.

Meditation really helped me (Headspace app) as well as keeping a journal which included five things I was grateful for each day. Gratitude really is a great natural mental booster and helps you even for a second to focus on whats good.

Cognitive Behavioural Therapy exercises also helped me stop my mind from spiralling. If youve never had CBT before dont worry, a great book I read called Feeling Good, the new mood therapy is full of easy to follow CBT exercises that you can use to talk back to the destructive thoughts that are coming out to play.

The main advice I can give to you is TALK!

Even though you know isolation is transitory it will still mess with your head, and your mood could change from hour to hour so make sure youre talking it out.

I mean this with all sincerity, if theres no one in your world you feel you can talk too then drop me an email and I will send you my phone number so we can chat.

Im also a trained Samaritan volunteer so know how to give someone the space they need to chat, albeit its been a few years since I practised but genuinely happy to talk.

One day this will all be a distant memory but right now its a bit s**t so dont be afraid to reach out to those around you.

[emailprotected]

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Cork man shares hacks for isolation that stopped him from 'going insane' - Echo Live

Notice Regarding Business Alliance Between Sanbio and Ocumension in the Research, Development and Commercialization of Innovative Stem Cell Therapies…

TOKYO--(BUSINESS WIRE)-- SanBio Co., Ltd. (Keita Mori, Representative Director and President, hereinafter SanBio) (TOKYO:4592) hereby announces that SanBio has entered into a business alliance with Ocumension (Hong Kong) Limited (hereinafter Ocumension).

1. Overview of the business alliance

SanBio and Ocumension have entered into a business alliance for the research, development and commercialization of innovative stem cell therapies for ophthalmic diseases. Both companies will jointly develop SanBios proprietary modified mesenchymal stem cell medicines with an initial focus in retinitis pigmentosa and dry age-related macular degeneration (SB623 cells), and optic neuritis (MSC2 cells).

Ocumension brings together a highly experienced leadership team and a commitment to world-class ophthalmic drug development, said Keita Mori, CEO of SanBio. The strategic collaboration would enable SanBio to bring cutting-edge stem cell therapies to Greater China, where great unmet medical needs exist in ophthalmology.

Pursuant to the terms of the agreement, Ocumension will fund the initial investment of $6 million for the preclinical and manufacturing development, and the remaining preclinical and manufacturing development will be equally shared by both parties. Ocumension obtains exclusive rights from SanBio to develop and commercialize SB623 and MSC2 in Greater China (including Mainland China, Hong Kong, Macau and Taiwan; hereafter Territory) for ophthalmic indications. Ocumension will be responsible for all cost associated with clinical development and commercialization activities conducted in the Territory under the agreement. SanBio retains all rights for ophthalmic indications for the rest of the world, and all rights for non-ophthalmic indications globally. SanBio will be eligible to receive up to $71 million in milestone payments. In addition, Ocumension will pay SanBio tiered royalties from single digit to low teens as a percentage of annual net sales in the Territory.

Ocumension is focused on bringing novel therapeutics to the ophthalmology market, especially in China, and so we welcome the opportunity to partner with SanBio, one of the leading R&D companies in the cell therapy, to develop SB623 and MSC2, said Ye Liu, CEO of Ocumension. We believe that based on the novel neurologic mechanism, these two products will offer significant potential benefits for the patients who cannot be satisfied by the existing treatment.

2. Profile of the business alliance partner

(1)

Company name

Ocumension (Hong Kong) Limited

(2)

Headquarters

Shanghai

(3)

Representative

Ye Liu, CEO

(4)

Main business

Research and development of ophthalmic drugs

(5)

Capital

Not disclosed at the request of Ocumension.

(6)

Date established

March 7th, 2018

(7)

Major shareholder and shareholding ratio

Not disclosed at the request of Ocumension.

(8)

Relationships with partnering companies

Capital relationship

Not applicable

Personal relationship

Not applicable

Business relationship

Not applicable

Affiliated party as prescribed in the Rules of Corporate Accounting

Not applicable

(9)

Consolidated earnings and financial position for the last three fiscal years

Not disclosed at the request of Ocumension.

3. Schedule

(1)

Date of resolution at companys Board of Directors meeting

March 31, 2020

(2)

Conclusion date of the Agreement

March 31, 2020

4. Outlook

The SanBio Group is reviewing the impact of the agreements with Ocumension on its financial results for the fiscal year ending January 31, 2021 and shall disclose any matters that require disclosure without delay.

View source version on businesswire.com: https://www.businesswire.com/news/home/20200331005923/en/

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Notice Regarding Business Alliance Between Sanbio and Ocumension in the Research, Development and Commercialization of Innovative Stem Cell Therapies...

Designer, injectable gels to prevent transplanted Schwann cell loss during spinal cord injury therapy – Science Advances

INTRODUCTION

Spinal cord injury (SCI) is a debilitating condition that currently has no regenerative-based therapy available in the clinic. SCI results in substantial financial, physical, and emotional burdens for patients and their families. Cell-based therapies have emerged as a promising approach to encourage regeneration and functional recovery after SCI (1). The transplantation of autologous human Schwann cells (SCs) is currently being investigated in U.S. Food and Drug Administration clinical trials for patients with thoracic and cervical level SCI (14). SCs are glial cells found in the peripheral nervous system that promote axon regeneration after peripheral nerve injury. Over the past two decades, several preclinical studies have demonstrated that SCs have tremendous potential to promote regeneration after SCI when delivered directly into the lesioned cavity that forms after injury (4, 5). Compared to possible SCI regenerative therapies with pluripotent cells, SCs are highly pure, well characterized, and relatively easy to isolate and expand from the patients sural nerves (2). In addition, the autologous nature of patient-derived SCs removes the need for immunosuppressant drugs compared to allogeneic cell therapies (6).

Unfortunately, direct local injection of SCs into the SCI results in significant transplanted cell loss and death (7). During injection, typically up to 60% of cells fail to even reach the target site (8). This may be due to a combination of factors, including cell membrane damage during injection and cell reflux out of the spinal cord (810). In addition, previous studies have shown that significant transplanted cell death rapidly occurs as early as 10 min after injection, leading to poor long-term SC survival. Typically, only ~20% of cells survive at 1 week and less than ~5% of transplanted SCs survive 1 month after transplantation, which may correlate with the often mixed functional results observed with SC therapies (7, 8, 11, 12). Because SC retention has been correlated with decreased SCI cavitation and symptomatic relief (5, 13), we hypothesized that increasing the number of transplanted viable cells early after delivery would markedly improve therapeutic efficacy. However, due to limited transplantation volume in the SCI lesion, simply increasing the number of injected cells is not feasible.

In this study, we sought to use a bioengineered injectable material strategy to address three key challenges that hinder SC survival during SCI transplantation (Fig. 1A). During cell delivery, cell loss is caused by (i) cell membrane damage due to extensional forces during injection and (ii) cell dispersal caused by reflux and leakage out of the SCI lesion. Shortly after injection, cell survival is further challenged by the (iii) lack of extracellular matrix (ECM) within the lesion, leading to anoikis, i.e., apoptosis of the anchorage-dependent SCs (14). Thus, for transplanted cells to be able to interact with the surrounding endogenous tissue and promote long-term repair, the cells must first overcome these three critical challenges (5). Here, we report a shear-thinning, injectable hydrogel that rapidly self-heals to significantly improve the survival and therapeutic function of transplanted SCs as a combination therapy for the treatment of SCI.

(A) Schematic depicting three key challenges SCs face during the transplantation process: (i) membrane damage from extensional forces exerted during syringe needle injection, (ii) cell extrusion and dispersal from the SCI cavity injection site, and (iii) apoptosis due to loss of endogenous matrix. (B) SHIELD, an injectable gel designed to address these challenges in cell delivery. Component 1 is C7, a recombinant, engineered protein composed of seven repeats of CC43 WW domains (denoted as C) separated by hydrophilic spacers containing cell-adhesive peptides. Component 2 is a multi-arm, peptide copolymer, 8-armed PEG tethered with proline-rich peptides (denoted as P) and the thermoresponsive polymer (PNIPAM). (C) Amino acid sequences of SHIELD components: CC43 WW domain (C), proline-rich peptide (P), and the different cell-adhesive spacer variants. (D) Shear storage moduli of soft, medium, and stiff SHIELD formulations with RGD, RDG, YIGSR, and IKVAV cell-adhesive spacers. Data are means SEM. ****P < 0.0001 statistical significance, Tukey post hoc test. n = 3 to 8.

Three distinct features were designed into our hydrogel materials to address the three critical challenges of transplanted cell survival described above: (i) thixotropy to protect the cell membrane from damage during injection, (ii) rapid self-healing and stiffening in situ to localize the cells within the SCI lesion, and (iii) cell-adhesive ligands to promote SC attachment and spreading. Previous reports by us and others have demonstrated that thixotropic hydrogels that undergo plug-flow fluid mechanics can shield encapsulated cells from damaging mechanical forces during injection (9, 15, 16). Furthermore, we reasoned that a thixotropic, physical hydrogel would facilitate ease of surgical use, since cells can be pre-encapsulated within the gel and then injected at any time by simply applying a critical yield stress to induce flow. In contrast, previous work with fibrin-based gels and other chemically cross-linked gels require careful timing of the injection procedure to ensure that the material does not prematurely form a gel and clog the injection device (1719). Here, we designed a family of injectable, physical hydrogels for SC transplantation based on our previously reported SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) strategy (15). These materials are composed of two components that engage in two stages of cross-linking. The first stage occurs ex situ in which physical cross-links are formed between an engineered recombinant protein (C7) and a multi-arm, polyethylene glycol (PEG)poly(N-isopropylacrylamide) (PNIPAM) copolymer conjugated with proline-rich peptides (P) (Fig. 1, B and C, and fig. S1). These two components assemble ex situ via reversible, heterodimeric binding of two peptide domains (a CC43 WW domain and a proline-rich peptide) to form a weak gel with encapsulated SC (20, 21). When the gel is subjected to a force, the peptide-peptide bonds are disrupted, allowing the material to shear-thin and flow as a liquid. After the force is removed, the peptide-peptide bonds rapidly reform, allowing the gel to quickly self-heal.

The second stage of cross-linking occurs in situ to stabilize and stiffen the gel after injection, thereby preventing cell extrusion and leakage out of the lesion, a common challenge for therapies delivered in saline. At body temperature, which is above the PNIPAM lower critical solution temperature of 32C, the polymer undergoes hydrophobic collapse to provide secondary physical cross-linking to stiffen and reinforce the hydrogel network (Fig. 1B) (15). The ideal gel stiffness to retain viable, transplanted SCs within the SCI lesion is currently unknown. Therefore, we formulated materials with a range of the thermoresponsive PNIPAM polymer [0, 1.25, and 2.5 weight % (wt %)], resulting in soft, medium, and stiff gels, respectively (plateau shear moduli, G, ~5, 160, and 400 Pa; Fig. 1D and table S1). These materials span the approximate range of reported neural tissue stiffnesses for the SCI lesion (22).

SCs are an anchorage-dependent cell type and undergo anoikis when transplanted into an SCI lesion that lacks adhesive ECM proteins. Thus, to promote long-term SC viability, three different cell-adhesive ligands were designed to the spacer domain of the C7 protein (Fig. 1C). Previously, we reported a SHIELD variant that included the fibronectin-derived RGD cell-adhesive ligand for use in culture of human adiposederived stem cells (15). The RGD epitope is also known to promote SC adhesion and migration; therefore, this same variant was included here (23). In addition, laminin is known to be a major component of the neural ECM and is reported to promote SC elongation and migration through F-actin polymerization (2426). Therefore, C7 variants including the laminin-derived cell-adhesive ligands known to induce cell spreading and attachment, YIGSR and IKVAV (27, 28), were newly designed and synthesized. To incorporate these ligands into our C7 recombinant proteins, engineered plasmid cassettes were inserted into pET-15b plasmids and transformed into BL21(DE3) pLysS Escherichia coli. Proteins were expressed under the T7 promoter, purified by affinity column, and characterized by gel electrophoresis, amino acid analysis, and Western blotting (figs. S2, S3, and S4). As a negative control material, we also cloned, expressed, and purified a C7 variant containing the noncell-adhesive RDG sequence. As expected, modification of the cell-binding domain did not significantly alter the mechanical properties of the gel (Fig. 1C and fig. S5).

Cells can experience membrane damage when exposed to extensional forces during syringe needle injection (Fig. 1A) (9). This is hypothesized to occur even at the very slow flow rates used here for SCI transplantation (500 nl/min), since the magnitude change in linear velocity of the solution is governed by the geometry of the syringe needle device. As the cells pass from the syringe barrel [inner diameter (ID) = 0.485 mm] into an ultra-small needle (33 gauge, ID = 0.11 mm), the fluid linear velocity increases 20-fold. Previously, we have reported that shear-thinning, self-healing hydrogels can protect many different cell types from membrane damage during syringe needle injection (15, 29, 30). To evaluate the ability of our designed SHIELD material to protect SCs from cell membrane damage during transplantation, we recreated the injection procedure in vitro using the same injection parameters described in in vivo transplantation studies (Materials and Methods). As expected, when cells are delivered either in saline or a viscous solution of the C7 RGD polymer (5 wt %; fig. S6), a statistically significant fraction of the cells experience acute membrane damage (25 and 20%, respectively, compared to 3% for the no injection control; Fig. 2, A and B). Mixing the C7 RGD polymer with the PEG-P-PNIPAM polymer (both at 5 wt % final concentration) enables creation of a shear-thinning, rapidly self-healing hydrogel (Fig. 2C). As expected, changing the identity of the cell-adhesive ligand or the amount of PNIPAM within the gel formulation did not alter the shear-thinning gel behavior (Fig. 2C and fig. S7). Pre-encapsulating the SCs within these SHIELD variants provided significant cell membrane protection during syringe needle flow (Fig. 2, A and D). Soft, medium, and stiff SHIELD variants all resulted in statistically higher levels of cell protection compared to cell delivery in saline. Similarly, the presence or absence of the RGD cell-adhesive domain within the SHIELD material did not affect the cell membrane protection. Together with the observation that C7 RGD polymer alone is unable to provide protection, these data suggest that the shear-thinning/self-healing properties of the gel (and not the gel cell-adhesive properties) are responsible for the cell membrane protection. To further confirm this idea, SCs were delivered within a comparison hydrogel formed from decellularized basement membrane matrix of a murine Engelbreth-Holm-Swarm sarcoma (commonly known by the trade name Matrigel). As expected, this control hydrogel did not provide statistically significant cell membrane protection (fig. S8).

(A) LIVE/DEAD staining confirms that SCs experience significant membrane damage upon exposure to extensional forces as they travel through a syringe needle in saline at 500 nl/min. Green, calcein-AM (live cells); red, ethidium homodimer-1 (dead cells). (B) Percentage of SCs with membrane damage when injected in SHIELD gels is significantly lower than those injected in saline or C7 alone, regardless of SHIELD stiffness or cell-adhesive ligand. Data are means SEM.*P < 0.05, one-way analysis of variance (ANOVA) with Tukey post hoc test. n = 3 to 16 individual injections, three to seven independent experiments. (C) Shear-thinning and self-healing behavior of soft, medium, and stiff SHIELD gels with RGD cell-adhesive ligand and soft RDG SHIELD at 0.1 (7) and 10 (gray) Hz. Data are means SD. n = 3. (D) Representative LIVE/DEAD images of SCs injected at 500 nl/min in a viscous solution of C7 RGD alone; soft, medium, or stiff SHIELD gels with RGD ligands; or soft RDG SHIELD gel.

As SHIELD materials of low, medium, and high stiffness all provided cell membrane protection during injection, these materials were all further evaluated for their ability to support SC culture in vitro. SCs have been reported to respond to the mechanical stiffness of 2D culture substrates by altering their proliferation rate (31); therefore, we first evaluated proliferation in our 3D gels by quantifying total DNA over time. SCs were homogeneously encapsulated as individual, dissociated cells at a density of 1.5 107 cells ml1 gel for each cell-adhesive variant (fig. S9). In our stiffest RGD-containing SHIELD formulation, SC DNA content over 3 and 7 days was significantly reduced compared to cells in soft or medium stiffness gels (Fig. 3A). Therefore, this stiffest formulation was excluded from further studies. While DNA content was highest in the medium stiffness RGD SHIELD gel at both 3 and 7 days, the overall SC viability was similarly high (>80%) across all three gel stiffness variants, suggesting that this stiffness range can support SC viability within a three-dimensional (3D) hydrogel context (Fig. 3, A to D, and figs. S9 and S10). Similarly, SC viability was not significantly affected by the choice of cell-adhesive ligand across the entire range of gel stiffness, although complete lack of a ligand (the RDG SHIELD variant) was beginning to trend down in viability at day 7 for soft gels, consistent with the possible initiation of anoikis (Fig. 3, A to D, and fig. S10). Previous reports by others have suggested that SC elongation and migration are dependent on F-actin polymerization, both in vitro and in vivo during regeneration of the peripheral nerve (25, 26). Thus, an ideal hydrogel formulation would promote migration and elongated morphology of transplanted SCs after transplantation. Therefore, we selected to evaluate F-actin polymerization in the different cell-adhesive gel formulations via phalloidin staining. SCs in the RGD SHIELD gels extended significantly longer processes than cells in the RDG, YIGSR, and IKVAV SHIELD gels (Fig. 3, E and F). This SC morphology is an indicator of cell-adhesive ligand engagement with the RGD peptide. While cell processes were observed to extend from SCs in other formulations, these were predominantly found within large cell clusters, potentially indicating a preference for cell-cell contacts and/or self-secreted matrix, rather than interaction with the SHIELD ligands (Fig. 3, E and G). Across all cell-adhesive ligand variants, no statistically significant differences in SC process extension or cell cluster size were observed when comparing between the soft and medium stiffness gels (Fig. 3, E to G). Together, our in vitro SC analysis resulted in the selection of the medium stiffness SHIELD gel with the RGD ligand for continued study. In this SHIELD formulation, SC morphology and expression of normal SC markers P75 and S100 were maintained, supporting its further evaluation as an SC delivery vehicle in a preclinical model of SCI (fig. S9).

(A) DNA quantification of SCs encapsulated in soft, medium, and stiff RGD SHIELD gels for 3 and 7 days normalized to soft SHIELD gels. Data are means SEM. *P < 0.05, one-way ANOVA with Tukey post hoc test. n = 5 to 6, three independent experiments. (B) Representative LIVE/DEAD images of SCs encapsulated in medium stiffness SHIELD gels with varying cell-adhesive ligands after 3 days in culture. Green, calcein-AM (live cells); red, ethidium homodimer-1 (dead cells). (C and D) SCs display high viability (>80%) across all SHIELD cell-adhesive variants for both soft and medium stiffness gels after 3 days of culture, while at 7 days, SCs in soft SHIELD variants lacking a cell-adhesive ligand (RDG) show increased variability in viability. Data are means SEM. No statistical significance, one-way ANOVA with Tukey post hoc test. n = 2 independent experiments, each with 2 technical replicates. (E) Representative fluorescent images of SC morphology in medium stiffness SHIELD gels with varying cell-adhesive ligand domains after 3 days in culture. Green, phalloidin (F-actin); blue, DAPI (4,6-diamidino-2-phenylindole dihydrochloride; nuclei). (F) Quantification of SC process length in soft and medium stiffness SHIELD gels with varying cell-adhesive ligands demonstrates that RGD gels promote significantly longer SC cytoplasmic processes after 3 days in culture. Data are means SD. *P < 0.05, one-way ANOVA with Tukey post hoc test. n = 3 to 11, 2 independent experiments. (G) Quantification of SC cluster area in soft and medium stiffness SHIELD gels with cell-adhesive variants after 3 days demonstrates significantly larger cell clusters in the RDG, YIGSR, and IKVAV variants. Data are box and whisker plots with mean, min, and max. *P < 0.05, one-way ANOVA with Tukey post hoc test. n = 2 independent experiments, each with 2 technical replicates.

We selected a unilateral, cervical contusion SCI model in female Fischer 344 rats chosen to represent the most commonly encountered SCI in patients (32). Briefly, a right, 75-kdyne contusion injury was performed at the fifth cervical (C5) level following a dorsal laminectomy (Fig. 4A). After 2 weeks of recovery, which is considered the subacute phase for this SCI model (33), an intended dose of 4.0 105 SCs was delivered in either SHIELD (n = 13) or saline (n = 13) using a 33-gauge Hamilton syringe (table S2). Injury only (n = 8) and saline (n = 8) injection served as negative controls. Transplanted SC retention was assessed at 48 hours (N = 10) and 4 weeks (N = 16) after transplantation. Consistent with our in vitro transplantation model data, we observed significantly higher numbers of P75+ cells at the lesion site when delivered in SHIELD compared to saline (Fig. 4D). Transplantation of exogenous, syngeneic SCs was confirmed by labeling cells with Qtracker655 and costaining for P75 (fig. S11), and control animals exhibited no P75+ staining (fig. S12). At 48 hours, 32,000 7700 viable, transplanted cells were observed in animals receiving SHIELD, while 4300 2500 viable, transplanted cells were found in animals receiving saline. Thus, while both cohorts had markedly fewer cells than the initial cell dosage, SHIELD transplantation resulted in a ~740% increase in viable local cell delivery compared to the clinical standard of saline transplantation. At 4 weeks, 29,000 8000 viable cells delivered with SHIELD were observed, while 2400 800 viable cells were counted for the saline-delivered animals, a ~10-fold increase in local delivery of viable cells compared to saline. In spinal cord cross sections at both time points, larger SC transplants were observed for SHIELD delivery compared to saline delivery (Fig. 4E). Consistent with reports by others, in some cross sections, the track left by the injection needle is still visible, suggesting that the reflux of saline during needle removal can result in a significant loss of transplanted cells (fig. S13). Together with our in vitro data, these observations suggest that the use of a shear-thinning, self-healing hydrogel can significantly improve SC delivery to the SCI lesion by providing cell membrane protection during injection and limiting cell extrusion from the injection site into the subarachnoid space.

(A) Schematic of SCI model. Following a C5 laminectomy, Fischer rats receive a right 75-kdyne contusion injury. Two weeks later (subacute phase), SCs are transplanted directly at the injury site using an automated syringe needle device. (B) Experimental time course of in vivo model of SC transplantation, behavioral assessment, and tissue harvest. SC retention is assessed in spinal cord explants at 48 hours and 4 weeks after transplantation. Behavioral assessments of forearm function are performed before injury, after injury, and weekly following SC transplantation. (C) Quantification of the number of P75+ SCs at 48 hours and 4 weeks after transplantation demonstrates significantly higher SC retention when delivered in SHIELD compared to saline. Data are means SEM. **P < 0.01 and *P < 0.05, unpaired, two-tailed t test. n = 8 animals, N = 2 independent experimental replicates. (D) Representative fluorescent images of P75+ cells in spinal cord explants transplanted with SCs in saline or SHIELD at 48 hours and 4 weeks after transplantation.

To assess the ability of our SHIELD material to promote cell adhesion in vivo, analysis of SC transplant morphology was performed at 48 hours. Consistent with reports by others, cells delivered in saline remained as compact clusters of spherical cells, indicative of the loss of native matrix within the SCI lesion. In contrast, SCs delivered in SHIELD were significantly more elongated and extended throughout the lesion (Fig. 5A), indicative of cell adhesion to the transplanted material. Cell shape was quantified by the spindle ratio defined as the major axis length divided by the short axis length. Cells in SHIELD had a statistically significant 3.05-fold increase in spindle ratio compared to cells in saline (Fig. 5B). At 4 weeks after transplant, cells delivered in saline were primarily observed near the original site of injection at the C5 level of the spinal cord (Fig. 5C, top). In contrast, SCs delivered in SHIELD were found to extend longitudinally through the cord, in both the rostral and caudal directions (Fig. 5C, bottom). Transplanted cell distribution was quantified by manually counting the number of cells within a 60-m-thick section at 360-m intervals. Consistent with our qualitative observations, cells delivered in SHIELD were significantly more spread throughout the cord than those in saline (P = 0.0013, Kolmogorov-Smirnov analysis; Fig. 5D).

(A) Fluorescent images of P75+ SCs 48 hours after transplantation demonstrate significant morphological differences between cells delivered in saline compared to SHIELD. (B) Quantification of SC morphology as measured by spindle ratio (major axis/minor axis) demonstrates that SHIELD-delivered SCs are substantially more elongated than saline-delivered cells. Data are means SEM. *P < 0.05, unpaired t test. n = 4 to 5 animals, N = 2 independent experimental replicates. (C) Distribution of SCs within the spinal cord 4 weeks after transplantation was visualized in histological cross sections as P75+ areas and projected onto the C3 to C7 schematic. SHIELD-transplanted SCs show a greater spatial distribution within the endogenous tissue compared to saline. (D) Histogram of P75+ SCs observed throughout the spinal cord within different segments 4 weeks after transplantation shows significantly greater distribution in both the rostral and caudal directions for SHIELD-delivered cells compared to saline. Data are means SEM. *P < 0.05, Kolmogorov-Smirnov test of cumulative distribution. n = 4 to 5 animals, N = 2 independent experimental replicates.

It has long been hypothesized that the mechanism of SC action on SCI regeneration and functional recovery occurs through modulating the secondary injury response within the endogenous tissue, resulting in minimization of cystic cavitation (5). Unfortunately, poor transplanted cell survival limits their therapeutic potential to enact this response. Here, we immunostained spinal cord sections with endogenous tissue and cell markers to assess the secondary injury and native tissue response, including the volume of the cystic cavity. To assess the secondary injury that occurs following SCI, we defined the area devoid of cell nuclei and astrocytes as the cystic cavity, the adjacent 100 m as the lesion, and the subsequent adjacent 100 m as the peri-lesion (Fig. 6A) (34). The total cavitation volume was quantified by measuring the cavity cross-sectional area at 360-m intervals throughout the entire lesion. Delivering SCs in SHIELD significantly reduced the cystic cavitation volume compared to animals treated with SCs in saline, saline only, and injury only (P = 0.004, 0.014, and 0.0329, respectively; Fig. 6B). When comparing the cystic lesion volume in each individual animal to their number of surviving transplanted SCs at 4 weeks (data in Fig. 4C), a statistically significant correlation was observed (P = 0.0314, Pearson correlation test, 95% confidence interval). These data are consistent with previously published reports that demonstrate transplanted SCs result in greater tissue sparing (5). The glial scar response was quantified by measuring the percentage of area staining positively for reactive astrocytes [glial fibrillary acidic protein (GFAP)] in the adjacent lesion and peri-lesion regions. We found that while all groups had formation of a glial scar, SHIELD delivery of SCs led to a significant reduction in the reactive astrocyte response in the peri-lesion space (Fig. 6C). To quantify the neuronal population within the spinal cord after injury, we performed III-tubulin staining and compared the staining intensity within the injured and uninjured hemispheres (excluding the cavity) to account for interanimal variability (Fig. 6A). While injury only and saline only control animals had a ~0.6 neuronal ratio between the injured and uninjured hemispheres, delivery of SCs in SHIELD had a significantly higher neuronal ratio of ~0.8 (P = 0.0487 and 0.0426, respectively; Fig. 6D). This result is consistent with previous reports in which increased SC retention was observed with increased neuronal and axon area (13). Because of the timing of this staining (4 weeks), this ratio is likely a measurement of neuronal and axonal sparing and minimization of the secondary injury, rather than axonal regeneration.

(A) Schematic of secondary injury characterization in spinal cord sections delineating the cystic cavitation, the adjacent lesion and peri-lesion areas, and uninjured and injured hemispheres. (B) Representative whole scan fluorescent images of spinal cord sections display cavity areas across all groups. Cyan, GFAP. Quantification of cavitation volume shows significantly smaller cavities in animals treated with SCs delivered in SHIELD. Data are means SEM. *P < 0.05, one-way ANOVA, unpaired t test post hoc analysis. n = 7 to 8, N = 2 independent experiments. (C) Glial scar formation is a hallmark of SCI and can be observed by increased GFAP staining surrounding the cystic cavity. Cyan, GFAP. Quantification of GFAP-positive area across all groups reveals a decrease in the peri-lesion region of SHIELD-delivered SCs compared to saline only injection. Data are means SEM. *P < 0.05, one-way ANOVA with Tukey post hoc test. n = 5 to 6, N = 2 independent experiments. (D) Representative fluorescent images of III-tubulinstained spinal cord explants reveal diminished neuronal presence in the injured hemisphere. Normalized injured to uninjured III-tubulin intensity demonstrates significantly increased neuronal staining in animals treated with SCs in SHIELD compared to injury only and saline-injected groups. Data are means SEM. *P < 0.05, one-way ANOVA with Tukey post hoc test. n = 3, N = 2 independent experiments.

We also examined the response of endogenous immune and vascular cells to SC transplantation in saline or SHIELD (Fig. 7). Here, we only observed a significant difference in pan-macrophage (ED1) response in the lesion and peri-lesion areas, where SC delivery in SHIELD appeared to have fewer macrophages within the injury after 4 weeks compared to injury only control animals (Fig. 7A). SCs are known to influence macrophage behavior through paracrine signaling (35), and thus, improving the localized survival of SCs has the potential to influence numerous downstream cell signaling processes. However, as we only observe hematogenous macrophage response at this single 4-week time point, future work would need to include multiple time points and cellular identities to characterize the full dynamic response of immune cell infiltration, as much of the early inflammation is often resolved by 4 weeks (36, 37). No differences in lesion and peri-lesion staining were observed for microglia (Iba1/Tomato lectin) and vascularization (Tomato lectin) (Fig. 7, B and C). Similarly, no significant differences were observed for each stain (ED1, Iba1, and Tomato lectin) when comparing between the injured and uninjured hemispheres (fig. S14). Together, these results support the idea that increased delivery of SCs to the site of an SCI contusion lesion modulates the secondary injury, resulting in decreased formation of a cystic cavity.

(A) Representative fluorescent images of immunostained spinal cord sections for pan-macrophage marker, ED1. Quantification of ED1+ area revealed statistically decreased staining in the lesion and peri-lesion regions for animals receiving SCs in SHIELD compared to injury only controls. Data are means SEM. *P < 0.05, one-way ANOVA with Tukey post hoc test. n = 7 to 8, N = 2 independent experiments. (B and C) Representative fluorescent images of immunostained spinal cord sections for microglia (Iba1) and vasculature (Tomato lectin). Quantification revealed no significant differences in (B) Iba1 and (C) Tomato lectin staining in the lesion and peri-lesion regions across the groups. Data are means SEM. n = 3 to 8, N = 2 independent experiments. NS, not significant.

SCs have been a therapeutic focus for treating SCI for several decades. Unfortunately, while SCs have been demonstrated to limit cystic cavitation in several preclinical models, the reported functional improvement in these models has often been modest, which we hypothesized to be due to poor viable cell delivery. Therefore, we evaluated the combination cell and injectable hydrogel therapy on functional forelimb recovery. Both motor and sensorimotor tests were performed to obtain a more complete picture of right forelimb function, as contusive SCI can result in both motor and sensory deficits (32, 38). To assess forelimb motor recovery, we measured forelimb grip strength (39, 40). Both combined and right arm strength were evaluated to assess whether left arm compensation influences the right arm behavior. As anticipated, unilateral C5 contusion results in both combined and right forelimb grip strength deficit, although the reduction is significantly greater in right forearm only tests (Fig. 8, A and B). Over the experimental period, no group reached pre-injury level; however, in both combined and right forelimbs, we observed a significant increase in grip strength for animals treated with SCs in SHIELD compared to injury only controls (P = 0.0295 and 0.0114, respectively; Fig. 8, A and B). Furthermore, right forelimb grip strength for SCs in SHIELD-treated animals was significantly higher than saline only and SCs in saline (P = 0.0138 and 0.0165, respectively; Fig. 8, A and B). No significant improvement was observed for the animals treated with SCs in saline. We also assessed the sensorimotor recovery of animals through the Horizontal Ladder Walk test (41). This assay measures forelimb coordination by counting the number of forearm missed steps (complete and partial) as the animal traverses a horizontal ladder with unevenly spaced rungs. As anticipated, we observed a significant increase in missed steps after cervical contusion SCI (Fig. 8C). At 4 weeks after transplant, animals treated with SCs in SHIELD saw a significant decrease in missed steps compared to injury and saline only animals. As with the forelimb strength test, no significant improvement was observed for animals treated with SCs in saline (P = 0.713; Fig. 8C). Furthermore, at 4 weeks after transplant, animals treated with SCs in SHIELD only missed 16.62 1.63% of steps, which was a significant improvement compared to their post-injury behavior (35.27 1.77%, P = 0.0038) and statistically similar to their pre-injury behavior (13.81 0.06%, P = 0.970). In comparison, animals with injury only, saline only treatment, or transplantation of SCs in saline had statistically negligible improvement from their post-injury behavior (P = 0.966, 0.723, and 0.373, respectively). When comparing the functional outcome in each individual animal to their number of surviving transplanted SCs at 4 weeks (data in Fig. 4C), a statistically significant positive correlation was observed between the SC count and the combined forelimb grip strength, while a negative correlation was observed between the SC count and the percentage of missed steps in the horizontal ladder assay (P = 0.0135 and 0.0068, respectively, Pearson correlation test, 95% confidence interval). No statistically significant correlation was observed between SC count and right forelimb grip strength (P = 0.1207), likely due to the high individual variability in the right grip strength measurement. Together, these data are consistent with previously published reports and support our hypothesis that an improvement in the number of successfully transplanted SCs can improve therapeutic outcomes (5).

Forearm motor and sensorimotor function was assessed before injury, after injury, and weekly after transplantation using a grip strength meter (A and B) and Horizontal Ladder Walk test (C), respectively. (A) Combined forearm grip strength significantly decreases following SCI. By 4 weeks after transplantation, grip strength was significantly increased in animals treated with SCs in SHIELD compared to injury only animals. (B) Relative to combined forearm grip strength, quantification of right forearm strength alone results in a more pronounced deficit due to the right, unilateral contusion injury. However, similar to combined forearm strength, by 4 weeks, right forearm grip strength is significantly increased over injury only controls. Data are means SEM. ****P < 0.0001 and *P < 0.05, one-way ANOVA with Tukey post hoc test. n = 47 before injury, n = 53 after injury, n = 7 to 8 after transplantation, N = 2 independent experiments. (C) Forelimb coordination was assessed with the Horizontal Ladder Walk test; after injury, a decrease in coordination presents as an increase in missed steps. Four weeks following SC transplantation in SHIELD, a significant decrease in percentage of missed steps is observed compared to injury only control animals. In addition, SC delivery in SHIELD results in a similar percentage of missed steps to that of pre-injury levels. Data are means SEM. *P < 0.05 and $P = 0.970 comparison between before injury and 4-week SCs in SHIELD, one-way ANOVA with Tukey post hoc test. n = 37 before injury, n = 53 after injury, n = 7 to 8 after transplantation, N = 2 independent experiments.

Successful delivery of transplanted cells and maintenance of cell viability after transplantation are common challenges faced by many cell-based therapies (7, 17, 42, 43). As the dosage and retention of delivered viable cells have been correlated with symptomatic outcomes for a variety of injury and disease models, the inability to successfully transplant cells directly into damaged tissue is a significant hurdle for clinical translation of many potential regenerative medicine therapies (4246). Here, we specifically address the challenge of SC delivery for SCI through the design of a novel biomaterial strategy. SCs are currently in clinical trials for the treatment of SCI, owing to their ability to reduce the secondary injury response (cystic cavitation, glial scar formation, and neuronal loss) that occurs after injury in preclinical models. Unfortunately, due to limited survival of the transplanted cells and poor localization to the target site, SCs have yet to demonstrate their full potential in improving functional, behavioral recovery. Various approaches to combat transplanted cell death have been explored in the past, often focusing on the codelivery of drugs (i.e., soluble apoptosis inhibitors), growth factors, and/or genes (11, 47, 48). While potentially beneficial, this approach requires careful attention to the spatial and temporal delivery of the proper dosage of prosurvival factors to be successful. When the location, timing, and dosage are not precisely controlled and optimized, delivery of these factors has the potential to impede therapeutic efficacy and even, in some cases, cause further functional deficits (4951).

Therefore, to move cell-based therapies toward clinical translation, we proposed an alternative strategy that does not require the careful spatiotemporal dosing control that is associated with small molecules, growth factors, and gene therapies. Our approach improves cell survival by designing a combination cell-plus-hydrogel therapy that addresses three key underlying causes of cell loss: (i) membrane damage during injection, (ii) cell reflux away from the injection site, and (iii) anoikis due to loss of endogenous matrix. While other hydrogel materials have been studied to improve the retention of viable cells, these systems often only address a single cause of cell loss during transplantation and/or rely on the use of a decellularized matrix from Engelbreth-Holm-Swarm murine sarcoma (often known by the trade name Matrigel), which is not clinically translatable (13, 52, 53). Other natural-based materials have been investigated for improving SC transplantation, including an acellular, injectable peripheral nerve matrix; however, many of these materials suffer from batch-to-batch variability that can limit the reproducibility of the SC response (54). In contrast, our designed gel material, SHIELD, contains three mechanisms of action within a single material that results in a greater than 700% increase in transplanted SC survival compared to the clinical standard of saline delivery. In addition, the biomechanical and biochemical properties of SHIELD were designed to not only increase cell number but also improve transplanted cell function by providing a 3D, cell-adhesive, physical support with ECM cues selected to enhance F-actin polymerization and elongated cell morphology, which has been correlated with SC migration in vitro and in vivo during regeneration of the peripheral nerve (25, 26, 55). The use of SHIELD to improve the successful transplantation of SCs resulted in a significant reduction in the secondary injury and pronounced increases in forelimb strength and coordination after SCI, thereby demonstrating functional efficacy. Moreover, our designed material platform is fully chemically defined for rigorous reproducibility and future evaluation by regulatory agencies.

While we report a significant improvement in cell retention compared to the clinical standard of saline delivery, cell loss from initial injection number does occur using our material platform. This result indicates that additional optimization can be undertaken to further improve cell survival by using the modular design of the hydrogel system for a complete and systematic interrogation of combinations of biochemical ligands. As biochemical ligands can often have nonlinear synergistic and antagonistic effects, these future studies would likely require computational algorithms to assist in identifying the optimal ligand formulation, as has been previously reported for other cell types (56, 57). In addition, in the scope of this study, we report the use of a combination cell-plus-hydrogel therapy on spinal regeneration and functional recovery. However, further investigation of our designer, injectable hydrogel alone is of considerable interest, as material alone may potentially have endogenous effects, as reported for other injectable materials (58, 59). These future experimental opportunities for continued material optimization highlight the potential of our platform not only for use in SCI but also for numerous other clinical indications that suffer from similar limitations. Last, while our study was focused on the use of a designer hydrogel platform for SCs, as they are currently in clinical trials, our study has broader implications for use of other cell types in treating SCI. The modular nature of this hydrogel system would enable easy customization for alternative cell types and for other clinical indications beyond SCI where the successful transplantation of cell-based therapies is critical for therapeutic efficacy.

SHIELD component synthesis. SHIELD hydrogels are physically cross-linked, two-component hydrogel systems composed of (i) a recombinant engineered protein termed C7 and (ii) a synthetic 8-arm PEG polymer modified with proline-rich peptides (P) and the thermoresponsive polymer, PNIPAM. The recombinant C7 proteins were designed to contain seven repeats of the CC43 WW protein binding domain interspaced with six repeats of selected cell-adhesive domains (RGD, RDG, YIGSR, and IKVAV; see fig. S2 for full amino acid sequences) (20). C7s were expressed in BL21(DE3)pLysS E. coli (Life Technologies) based on a previously published procedure (21). Briefly, C7s were cloned into pET-15b plasmids and expressed under control of the T7 promoter. Bacteria containing the plasmids were cultured in Terrific broth to an OD600 (optical density at 600 nm) of 0.8, and expression was induced by 1 mM isopropyl -d-1-thiogalactopyranoside. After 24 hours, the bacteria were harvested by centrifugation, resuspended in lysis buffer [10 mM tris, 1 mM EDTA, and 100 mM NaCl (pH 8.0)], and lysed by multiple freeze-thaw cycles. Lysates were treated with deoxyribonuclease I and 1 mM phenylmethanesulfonyl fluoride protease inhibitor. The C7 variants were purified by affinity chromatography via the specific binding of an N-terminal polyhistidine tag to Ni-nitrilotriacetic acid resin (Qiagen), dialyzed against phosphate-buffered saline (PBS), and concentrated by diafiltration across Amicon Ultracel filter units (Millipore). Protein purity was confirmed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and amino acid sequencing. Expressed protein was separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, blocked with nonfat milk, and probed with 6HisTag (Cell Signaling Technology) primary antibody. The primary antibody was detected using horseradish peroxidaseconjugated secondary antibody (1:10,000; donkey anti-rabbit, Jackson ImmunoResearch). Blots were developed with SuperSignal West Pico or Femto Chemiluminescent Substrates (Pierce) and imaged using a ChemiDoc MP gel imaging system (Bio-Rad). Before use for any in vivo transplantation studies, C7 was endotoxin-purified using previously published protocols (60). Briefly, endotoxin was removed from the C7 solution using thermocycling in a 1% Triton X-114 solution. Triton X-114 was then removed from the solution using Bio-beads SM-2 Resin (Bio-Rad).

PEG-P and PEG-P-PNIPAM were synthesized according to previously published protocols (15, 30). Briefly, 8-arm PEG vinyl sulfone (8-arm PEG-VS) with a nominal molecular weight of 20 kDa was purchased from Nanocs (Boston, MA). Peptide P (EYPPYPPPPYPSGC, 1563 g/mol) was purchased through custom peptide synthesis from GenScript Corp. (Piscataway, NJ, USA). All other chemicals were purchased from Sigma-Aldrich (Milwaukee, WI) unless otherwise noted. PNIPAM endcapped with a thiol group (PNIPAM-SH) (molecular weight, 30 kDa) was synthesized using reversible additionfragmentation chain transfer polymerization and conjugated to the 8-arm PEG-VS via a Michael-type addition reaction according to previously published protocol. The stoichiometry of the PNIPAM conjugation reaction was altered to modify, on average, either 0.5 or 1 arm of the PEG-VS as confirmed by 1H nuclear magnetic resonance (NMR) (fig. S1). Unreacted arms of PEG-VS were further reacted with excess P peptide in the presence of tris(2-carboxyethyl)phosphine (Sigma-Aldrich, St. Louis, MO). The stoichiometry of the P conjugation reaction was altered to modify, on average, 7 arms of the PEG-VS as confirmed by 1H NMR (fig. S1). The PEG-P-PNIPAM copolymer solution was lyophilized, washed with chloroform to remove unreacted PEG, and then dialyzed (molecular weight cutoff, 3 kDa) against deionized water (pH 7.4) to remove unreacted PNIPAM and P. For comparison, PEG-P copolymer was synthesized by reacting 8-arm PEG-VS with excess P and purified as described above. Once again, the P conjugation reaction was confirmed by 1H NMR (fig. S1).

SHIELD gel fabrication. SHIELD gels were prepared by adding appropriate amount of PBS to reach a 10% (w/v) C7 and a 10% (w/v) PEG-P or PEG-P-PNIPAM solution. For gel fabrication, each WW domain in C7 was treated as one C unit, and each pendant P peptide group in the PEG-P-PNIPAM copolymer was treated as one P unit and components were mixed to achieve a C:P ratio of 1:1 and final polymer concentration of 10% (w/v). To fabricate the three different SHIELD mechanical formulations (soft, medium, and stiff), appropriate amounts of 10% (w/v) PEG-P and/or PEG-P-PNIPAM were added to the 10% (w/v) C7 solution for a final 10% polymer solution with 0, 1.25, and 2.5% final PNIPAM concentrations (table S1). The final concentration of cell-adhesive peptides in each hydrogel is estimated to be 6.5 mM.

SHIELD rheological characterization. Dynamic oscillatory rheology experiments were performed on a stress-controlled rheometer (AR-G2, TA Instruments, New Castle, DE) using a 25-mm-diameter cone plate geometry. Samples were loaded immediately onto the rheometer after mixing, and a humidity chamber was secured in place to prevent dehydration. Frequency sweeps from 0.1 to 10 Hz at 25 and 37C were performed at 1% constant strain to obtain storage moduli (G) and loss moduli (G). Shear-thinning and self-healing properties of the gel samples were characterized by measuring viscosity () under a time sweep mode at alternating low and high shear rates of 0.1 and 10 s1, respectively, for 60 s each and a total of 300 s.

Primary adult SCs were isolated from micro-dissected sciatic nerves of 8- to 10-week-old syngeneic female Fischer rats following a protocol approved by the Stanford Administrative Panel on Laboratory Animal Care. The National Institutes of Health (NIH) guidelines for the care and use of laboratory animals were observed (NIH publication no. 8523 Rev. 1985). SCs were expanded in growth medium [Dulbeccos modified Eagles medium (DMEM), fetal bovine serum, GlutaMAX, Antibiotic-Antimycotic (ABAM) (Life Technologies), pituitary extract (20 g/ml; Gibco), and 2 M forskolin (Sigma)] on poly-l-lysinecoated tissue culture plastic following a previously described protocol (fig. S8) (12). SCs were used between passages 2 and 4 for all in vitro and in vivo studies. To obtain pure (>90%) SC cultures from any contaminating endogenous endoneurial fibroblasts, SCs were separated on the AutoMACs (Miltenyi Biotec) using the Possel D2 software setting and the low-affinity nerve growth factor receptor p75 [supernatant 192-IgG (immunoglobulin G)] and mouse IgG magnetic microbeads (Miltenyi).

For in vitro cell injection studies, SCs were lifted by trypsinization, pelleted, resuspended, and counted. The SCs were pelleted again and resuspended in the different delivery materials to achieve a final cell density of 2.5 107 cells ml1. This cell density was selected to maximize the number of individual injection replicates that could be performed per independent round of study. SCs were encapsulated in 4 l of SHIELD hydrogels (soft, medium, and stiff formulations), C7 RGD alone, or Hanks balanced salt solution (HBSS; saline) and loaded into a 10-l Hamilton syringe. Cells were injected using the same in vivo injection parameters (33-gauge Hamilton syringe needle, 500 nl/min, 4-min rest) into a 24-well plate with 0.5 ml of SC media. After 30 min, cell viability was assessed by LIVE/DEAD staining (Life Technologies) following the manufacturers instructions and imaged on a Leica SPE confocal microscope.

For proliferation assays, SCs were lifted by trypsinization, pelleted, resuspended, and counted. SCs were pelleted again and resuspended in the 10% (w/v) C7 solution to achieve an initial cell seeding density of 1.5 107 cells ml1 in the final hydrogels. This cell density was selected to facilitate quantitative image analysis after 3 to 7 days of in vitro culture. Cell-containing C7 was mixed with PEG-P and/or PEG-P-PNIPAM at appropriate gel-forming ratios and transferred to 4 mm (D) 2.5 mm (H) silicone molds, covered with SC media, and incubated at 37C. After 3 and 7 days, media were removed, lysis buffer [20 mM tris-HCl, 150 mM NaCl, and 0.5% Triton X-100 (pH 7.4)] was added, and the gels were disrupted by agitation. DNA content was determined using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies), following the manufacturers instructions. Cytotoxicity at 3 and 7 days was also assessed by LIVE/DEAD staining and imaged on a Leica SPE confocal microscope.

For immunocytochemistry, SC-containing hydrogels were fixed with 4% paraformaldehyde in PBS at 37C for 30 min. Samples were permeabilized with PBS and 0.02% Triton X-100 (PBST) for 1 hour at room temperature and blocked with 5% bovine serum albumin and 10% goat serum (GS) in PBST for 1 hour at room temperature. The samples were then incubated with primary antibodies (rabbit P75 and mouse S100) diluted in PBST with 1% GS overnight at 4C. Samples were thoroughly washed with PBST and then incubated with Alexa Fluorconjugated secondary antibodies [AF488 goat anti-mouse, AF546 goat anti-rabbit (Life Technologies), or AF647 goat anti-rabbit (Life Technologies)] and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) as a nuclear counterstain for 4 hours at room temperature. For SC actin imaging, cells were incubated with fluorescein isothiocyanatephalloidin (1:1000; Sigma) and DAPI (1:500; Sigma) for 4 hours at room temperature after immunostaining. Samples were washed thoroughly with PBST and mounted using VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories). Samples were imaged using a Leica SPE confocal microscope.

Induction of cervical contusion SCI in rats. This protocol was approved by the Stanford Administrative Panel on Laboratory Animal Care. The NIH guidelines for the care and use of laboratory animals were observed (NIH publication no. 8523 Rev. 1985). Female Fischer 344 rats were used as animal models to determine the efficacy of delivering SCs within SHIELD for functional recovery after SCI. Numbers of animals per group were between 4 and 5 for 48 hours and between 8 and 10 for 4 weeks after transplantation based on (i) power analysis study to determine experimental numbers in animal models of SCIs to appropriately assess statistical differences and (ii) previous publications from the laboratory (table S2) (12). Transplantation studies were completed in two separate, independent studies to demonstrate reproducibility. Before surgery, animals were anesthetized with 1 to 3% isoflurane in oxygen, shaved around the neck and back region, and aseptically prepared with 70% ethanol and betadine solution. Lacri-lube Opthalmic Ointment was applied to the eyes to prevent drying, and the antibiotic penicillin was administered at 115 mU/kg at surgery for the subsequent 3 days. Analgesia (buprenorphine HCl, 0.14 mg/kg) was administered in saline by subcutaneous injection immediately after induction of anesthesia. During surgery, animals were kept on a heating pad to keep their body temperature as close to 37C as possible. All surgical equipment was sterilized by autoclaving for the first procedure, and for subsequent animals, all surgical equipment used was sterilized in a hot bead sterilizer. Incisions were made to the skin at the area around vertebral level C5, with the smallest incision possible to expose the cord. Muscle layers were separated using forceps to access the vertebra from C4-C6. A laminectomy was performed at the C5 vertebral level, with dura remaining intact, and the spinal column was stabilized with clamps. An Infinite Horizon (IH) device was used to provide a mild 75-kdyne contusion injury (32, 38). The IH impactor was positioned, calibrated over the right side of the exposed C5 spinal cord segment, and triggered to cause the unilateral cervical injury. Subsequently, the muscles were closed in layers (three stitches, double knot) using absorbable Vicryl-coated braided sutures size 5, and the skin incision was closed using clips. Subcutaneous saline was administered to prevent dehydration and normalize blood pressure. Clips will be removed once the wound has healed, usually between days 7 and 10 after injury.

Transplantation of SCs. On day 14 after injury, the same surgery was performed on all rats (as described above) to expose the contusion in the spinal cord. Rats were randomized into one of the following treatment groups: (i) injury only, (ii) saline only, (iii) SCs in saline, and (iv) SCs in SHIELD. Treatments were injected into the contusion site after bleeding has stopped. SCs were injected at 10 107 cells ml1 in a total volume of 4 l of HBSS (saline) or SHIELD using a 33-gauge needle attached to a 10-l Hamilton syringe, resulting in a final cell dosage of 4.5 105 cells, which is similar to previously published SC transplantation protocols (8, 12). Injections were performed using a microinjection apparatus (KOPF 995 stereotaxic apparatus and Stoelting Co, Quintessential Sterotaxic Injector) at a rate of 500 nl/min for more than 8 min. Saline only control injections were performed exactly as SC injections, with only 4 l of HBSS. Syringes were left in place for 4 min to minimize reflux after needle withdrawal. Afterward, the incision was closed in layers as described above, and subcutaneous saline was administered to prevent dehydration and normalize blood pressure. Following surgery, rats are returned to a new, clean cage with prewarmed paper towels.

Forty-eight hours or 4 weeks after SC transplantation, rats were terminally anesthetized using an intraperitoneal injection of euthanasia solution (sodium pentobarbital, Vedco) and perfused intracardially with approximately 200 to 300 ml of PBS followed by 200 to 300 ml of 4% buffered paraformaldehyde (PFA) solution. The perfusions were performed on the same day for all groups with the same fixative batch. Spinal cords were immediately explanted and post-fixed in PFA overnight at 4C. After fixation, cords were placed in 30% (w/v in PBS) sucrose overnight at 4C and then embedded in 10% (w/v in PBS) porcine gelatin (Sigma) blocks. Gelatin-embedded cords were then post-fixed with 4% PFA and placed in 30% sucrose overnight again. Tissue blocks were sectioned into 60-m sections using a freezing sledge microtome and stored in PBS until use.

Immunostaining of spinal cord explants was carried out between each of the groups, reducing differences in staining between days and groups. One in six sections from each spinal cord was analyzed for transplanted cell number, cystic cavitation, and endogenous secondary injury markers. Tissue sections were immunostained free-floating with mouse monoclonal or rabbit polyclonal antibodies. Sections were blocked with phosphate buffer containing 10% normal donkey serum and 0.2% (v/v) Triton X-100 for 1 hour at room temperature with shaking and then incubated for 48 hours at 4C in primary antibody diluted in blocking buffer. Mouse monoclonal antibodies used were (i) GFAP (1:500; Sigma) to identify astrocytes and unmyelinated SCs, (ii) Tuj1 (1:500; BioLegend) to stain for spared and regenerated axons, and (iii) ED1 (1:500; Bio-Rad, AbD Serotec) to stain for (hematogenous not resident) pan-macrophages. Rabbit polyclonal antibodies were used for (i) p75 (1:400; Promega, Madison, WI) and (ii) Iba1 (1:500; Wako) to stain for microglia and macrophages. DyLight 488conjugated Tomato lectin stain (1:100; Vector Laboratories) was used to stain for blood vessels and microglia. Following primary antibody staining, sections were washed three times with PBS and incubated with the appropriate secondary antibodies (donkey anti-rabbit and donkey anti-mouse, 1:400 in blocking buffer, Jackson ImmunoLabs) for 4 hours at room temperature. Last, sections were washed three times in PBS, mounted onto glass slides, and mounted in Diamond ProLong Gold Antifade mounting medium (Life Technologies) with DAPI. Low-magnification (10 objective) fluorescent images were taken using an IX70 Olympus microscope, and higher-magnification images (20 and 40) were taken using Leica SPE confocal microscope.

Tissue cavitation was measured in all animals to evaluate the effect of the transplanted SCs upon the secondary injury response and preservation of tissue at the lesion site. The combined area of any cystic cavities (including areas showing signs of degeneration, such as microcysts) throughout the serial cord was measured using ImageJ (34) from 4-week post-transplantation GFAP-stained tissue sections and multiplied by section thickness (60 m) to obtain an estimation of cavitation volume.

For calculation of transplanted SC retention, the total number of observed P75+ cells was manually counted using ImageJ in 48-hour and 4-week explanted sections, summed together, and multiplied by 6 (one of six serial sections). SC spindle ratio was determined by measuring the major and minor axis lengths of P75+ cells with clearly defined borders at the injection epicenter for all animals. SC distribution along the length of the cord and SCI lesion was determined by counting the number of P75+ cells at selected cross sections at the specified locations along the cord (1:6 stained serial sections, 60 m thick). To establish that P75+ cells were the result of transplantation and not endogenous migrating SCs, a subset of animals were transplanted with SC labeled with Qtracker655 (Invitrogen) using the manufacturers protocols (fig. S9). In vitro monitoring of Qtracker655-labeled cells confirmed coexpression of label and P75 for up to 4 weeks in vitro. In vivo, Qtracker655-labeled cells colocalized with P75+ cells at both 48 hours and 4 weeks after transplantation.

To evaluate the impact of SC retention on the expression of secondary injury response markers, one of six serial, 4-week post-transplantation sections were stained with GFAP (astrocytes), ED1 (pan-macrophages), Iba1 (microglia), and Tomato lectin (blood vessels and microglia). Tile scans of whole sections were taken at 20 magnification. Percent positive stained pixel area was measured using ImageJ in two regions of the sections: (i) the 100-m region adjacent to the cavity, defined as the lesion, and (ii) the subsequent 100-m region, defined as the peri-lesion (34).

Grip strength for behavioral assessment. Forelimb grip strength tests were performed on the animals before injury, after injury, and then weekly throughout the experimental timeline (4 weeks) to evaluate functional improvement of their forelimbs. For this test, animals were allowed to preferentially grip the handle bars of the grip strength machine (TSE Systems, 303500 Series) and pulled back until they let go of the bars, and the force with which they held on was recorded. This was repeated six times, for both forelimbs and right forelimb only, upon which the lowest and highest values were dropped and the remaining four values were averaged together.

Horizontal Ladder Walk for behavioral assessment. Horizontal Ladder Walk tests were performed on the animals before injury, after injury, and then weekly throughout the experimental timeline (4 weeks) to evaluate functional improvement of their forelimb coordination. For this test, animals were placed within a Plexiglas alleyway with metal rungs placed between 1 and 3 cm apart for varying difficulty. Animals were recorded (Sony HDR-CX675 camera) walking across the rungs, and the number of correct and incorrect (slipped and misplaced) forearm steps was recorded by blinded observers. The percentage of missed steps was calculated by dividing the number of incorrect steps by the total (correct and incorrect steps) number of steps and multiplying the quotient by 100.

All data are presented as mean SEM, and statistical analysis was performed using GraphPad Prism software. Statistical comparisons on shear moduli, cell viability, DNA content, SC cluster area, and SC process length were performed by one-way analysis of variance (ANOVA) with Tukey post hoc test. One-way ANOVA with Tukey post hoc test was performed on in vivo SC number and endogenous marker immunostaining and functional behavior results. Statistical comparisons were performed by unpaired, two-tailed t test with Welchs correction for in vivo SC spindle ratio. Statistical comparisons were performed by one-way ANOVA with unpaired, two-tailed t test for in vivo cystic cavitation. Correlations between measurements (transplanted SC count at 4 weeks, lesion volume, combined grip strength, right forelimb grip strength, and percentage of missed steps in horizontal ladder assay) were performed with a two-tailed Pearson correlation test with 95% confidence interval. Last, statistical comparisons were performed on in vivo SC distribution in the spinal cord by nonparametric Kolmogorov-Smirnov test for cumulative distribution analysis. Values were considered to be significantly different when the P value was less than 0.05.

Acknowledgments: We thank A. Foster and L. Cai for help with PNIPAM synthesis; K. Klett, S. Sanchez, and Eduardo Barrios for help with C7 expression; and C. Madl for discussions and expertise on 3D cell culture. This work was supported by the NIH (5R01EB027666, 1R21NS114549, 5R01HL142718, R01EB027171), Stanford Bio-X Interdisciplinary Initiative, Coulter Foundation, Wings for Life (WFL-US-020/14), Spinal Research (UK), and a seed grant from the Wu Tsai Neurosciences Institute at Stanford University. L.M.M. acknowledges a Geballe Laboratory for Advanced Materials Postdoctoral Fellowship and a Wu Tsai Neurosciences Institute Interdisciplinary Postdoctoral Fellowship. K.D. acknowledges a Stanford Bio-X Interdisciplinary Graduate Fellowship. Part of this work was performed at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-1542152. Spinal cord illustrations were adapted from Servier Medical Art, which is licensed under a Creative Commons Attribution 3.0 Unported License. Author contributions: L.M.M., G.W.P., and S.C.H. conceived the project. L.M.M., G.W.P., and S.C.H. designed the research. G.W.P. and S.C.H. supervised the research. L.M.M., V.M.D., A.T.W., K.D., G.W.P., and S.C.H. designed, performed, and analyzed all experiments. L.M.M. and R.A.S. cloned and expressed protein variants. L.M.M., V.M.D., K.D., and Z.A.M. performed all surgical procedures. L.M.M. and A.T.W. performed all immunofluorescence staining and imaging of histological samples. L.M.M. and M.J.K. performed cell culture experiments. All authors contributed to manuscript writing. Competing interests: S.C.H., W. Mulyasasmita, and L. Cai are inventors on a patent related to this work filed by the Board of Trustees of the Leland Stanford Junior University (no. 9399068, filed on 20 April 2015, published on 26 July 2016). The other authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Designer, injectable gels to prevent transplanted Schwann cell loss during spinal cord injury therapy - Science Advances

David Setboun Joins BrainStorm as Executive Vice President and Chief Operating Officer – GlobeNewswire

International Pharmaceutical Veteran to Lead Global Business and Commercial Development

Ralph Kern, MD, MHSc, Promoted to President

NEW YORK, April 01, 2020 (GLOBE NEWSWIRE) -- BrainStorm Cell Therapeutics, Inc. (NASDAQ: BCLI), a leading developer of cellular therapies for neurodegenerative diseases, today announced thatDavid Setboun, Pharm.D., MBA, has been appointed Executive Vice President and Chief Operating Officer.

International pharmaceutical executive Dr. Setboun, has directed commercial development, business strategy, and product launches for 2 decades at 3 major biopharmaceutical companies. Most recently, Dr. Setboun served as VP Corporate Development, Strategy & Business at Life Biosciences. In this role, David was instrumental in the development of various critical commercial, operating and funding milestones. From June 2015 to June 2018, he served as President, Biogen, France where he launched Biogens rare disease franchise. In addition, he supervised the launch of key neurology products and oversaw the Biosimilar business unit. Prior to his tenure at Biogen, Dr. Setboun, served as President, AstraZeneca, Portugal from 2012 to 2015, where he managed a product portfolio that grossed over $200 million annually. Prior to this role, David led the European Sales & Marketing function as AstraZenecas VP Europe where he directed a team of executives in marketing, commercial excellence, pricing and market access. During his tenure, he expanded AstraZenecas Oncology, Diabetes and Cardiovascular franchises. From 2002 to 2009, Dr. Setboun directed national and international teams and projects for Eli Lilly and Company in France and the USA. Fluent in French, English, Spanish and Portuguese, Dr. Setboun received his Pharmaceutical Doctorate (Pharm.D.) from University Paris XI in 1997 and his MBA from H.E.C Paris in 2001. More recently, David graduated from Harvard Business School (AMP 194).

BrainStorms investigational therapy, NurOwn is an amazing technology platform that has the potential to bring treatment to those with ALS and progressive MS and other neurodegenerative indications. With a fully enrolled Phase 3 Pivotal Trial for ALS in place, a Phase 2 Trial for progressive MS, an investigational platform in exosomes and the most ambitious biomarker program in ALS, I know I am joining a company with outstanding potential, said Dr. Setboun.

"Davids experience launching products for international biopharmaceutical companies and his expertise in commercial, business development and investments, make him an outstanding new member of the BrainStorm leadership team. His extensive experience at all levels of sales and marketing, coupled with his business strategy experience comes to us at a critical time in the evolution of NurOwn and helps us achieve our goal of bringing new treatment options to patients afflicted with neurodegenerative diseases, including ALS and progressive MS," saidChaim Lebovits, Chief Executive Officer ofBrainStorm.

Additionally, I am very pleased to announce Ralph Kern MD, MHSc, has been promoted to President of BrainStorm. Ralph joined BrainStorm in March 2017 as Chief Operating Officer and Chief Medical Officer. Directing our clinical development, Ralph has helped the Company rapidly achieve clinical milestone, after clinical milestone, while guiding the Company to expand its product pipeline to investigate additional applications and disease indications. In recognition of his ongoing, outstanding service, Ralph has been elevated to the position of President and Chief Medical Officer of BrainStorm.

About NurOwnNurOwn(autologous MSC-NTF cells) represent a promising investigational approach to targeting disease pathways important in neurodegenerative disorders. MSC-NTF cells are produced from autologous, bone marrow-derived mesenchymal stem cells (MSCs) that have been expanded and differentiated ex vivo. MSCs are converted into MSC-NTF cells by growing them under patented conditions that induce the cells to secrete high levels of neurotrophic factors. Autologous MSC-NTF cells can effectively deliver multiple NTFs and immunomodulatory cytokines directly to the site of damage to elicit a desired biological effect and ultimately slow or stabilize disease progression. NurOwn is currently being evaluated in a Phase 3 ALS randomized placebo-controlled trial and in a Phase 2 open-label multicenter trial in Progressive MS.

AboutBrainStorm Cell Therapeutics Inc.BrainStorm Cell Therapeutics Inc.is a leading developer of innovative autologous adult stem cell therapeutics for debilitating neurodegenerative diseases. The Company holds the rights to clinical development and commercialization of the NurOwnCellular Therapeutic Technology Platform used to produce autologous MSC-NTF cells through an exclusive, worldwide licensing agreement as well as through its own patents, patent applications and proprietary know-how. Autologous MSC-NTF cells have received Orphan Drug status designation from theU.S. Food and Drug Administration(U.S.FDA) and theEuropean Medicines Agency(EMA) in ALS. BrainStorm has fully enrolled the Phase 3 pivotal trial in ALS (NCT03280056), investigating repeat-administration of autologous MSC-NTF cells at six sites in theU.S., supported by a grant from theCalifornia Institute for Regenerative Medicine(CIRM CLIN2-0989). The pivotal study is intended to support a BLA filing for U.S.FDAapproval of autologous MSC-NTF cells in ALS. BrainStorm received U.S.FDAclearance to initiate a Phase 2 open-label multi-center trial of repeat intrathecal dosing of MSC-NTF cells in Progressive Multiple Sclerosis (NCT03799718) inDecember 2018and has been enrolling clinical trial participants sinceMarch 2019. For more information, visit the company'swebsite.

Safe-Harbor Statement

Statements in this announcement other than historical data and information, including statements regarding future clinical trial enrollment and data, constitute "forward-looking statements" and involve risks and uncertainties that could causeBrainStorm Cell Therapeutics Inc.'sactual results to differ materially from those stated or implied by such forward-looking statements. Terms and phrases such as "may", "should", "would", "could", "will", "expect", "likely", "believe", "plan", "estimate", "predict", "potential", and similar terms and phrases are intended to identify these forward-looking statements. The potential risks and uncertainties include, without limitation, BrainStorms need to raise additional capital, BrainStorms ability to continue as a going concern, regulatory approval of BrainStorms NurOwn treatment candidate, the success of BrainStorms product development programs and research, regulatory and personnel issues, development of a global market for our services, the ability to secure and maintain research institutions to conduct our clinical trials, the ability to generate significant revenue, the ability of BrainStorms NurOwn treatment candidate to achieve broad acceptance as a treatment option for ALS or other neurodegenerative diseases, BrainStorms ability to manufacture and commercialize the NurOwn treatment candidate, obtaining patents that provide meaningful protection, competition and market developments, BrainStorms ability to protect our intellectual property from infringement by third parties, heath reform legislation, demand for our services, currency exchange rates and product liability claims and litigation,; and other factors detailed in BrainStorm's annual report on Form 10-K and quarterly reports on Form 10-Q available athttp://www.sec.gov. These factors should be considered carefully, and readers should not place undue reliance on BrainStorm's forward-looking statements. The forward-looking statements contained in this press release are based on the beliefs, expectations and opinions of management as of the date of this press release. We do not assume any obligation to update forward-looking statements to reflect actual results or assumptions if circumstances or management's beliefs, expectations or opinions should change, unless otherwise required by law. Although we believe that the expectations reflected in the forward-looking statements are reasonable, we cannot guarantee future results, levels of activity, performance or achievements.

CONTACTS

Investor Relations:Preetam Shah, MBA, PhDChief Financial OfficerBrainStorm Cell Therapeutics Inc.Phone: + 1.862.397.1860pshah@brainstorm-cell.com

Media:Sean LeousWestwicke/ICR PRPhone: +1.646.677.1839sean.leous@icrinc.com

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David Setboun Joins BrainStorm as Executive Vice President and Chief Operating Officer - GlobeNewswire

Global Cell Therapy Technologies Market : Industry Analysis and Forecast (2019-2026) – Publicist360

Global Cell Therapy Technologies Marketwas valued US$ 12 billion in 2018 and is expected to reach US$ 35 billion by 2026, at CAGR of 12.14 %during forecast period.

REQUEST FOR FREE SAMPLE REPORT:https://www.maximizemarketresearch.com/request-sample/31531/

Global Cell Therapy Technologies Market

The objective of the report is to present comprehensive assessment projections with a suitable set of assumptions and methodology. The report helps in understanding Global Cell Therapy Technologies Market dynamics, structure by identifying and analyzing the market segments and projecting the global market size. Further, the report also focuses on the competitive analysis of key players by product, price, financial position, growth strategies, and regional presence. To understand the market dynamics and by region, the report has covered the PEST analysis by region and key economies across the globe, which are supposed to have an impact on market in forecast period. PORTERs analysis, and SVOR analysis of the market as well as detailed SWOT analysis of key players has been done to analyze their strategies. The report will to address all questions of shareholders to prioritize the efforts and investment in the near future to the emerging segment in the Global Cell Therapy Technologies Market.

Global Cell Therapy Technologies Market: Overview

Cell therapy is a transplantation of live human cells to replace or repair damaged tissue and/or cells. With the help of new technologies, limitless imagination, and innovative products, many different types of cells may be used as part of a therapy or treatment for different types of diseases and conditions. Celltherapy technologies plays key role in the practice of medicine such as old fashioned bone marrow transplants is replaced by Hematopoietic stem cell transplantation, capacity of cells in drug discovery. Cell therapy overlap with different therapies like, gene therapy, tissue engineering, cancer vaccines, regenerative medicine, and drug delivery. Establishment of cell banking facilities and production, storage, and characterization of cells are increasing volumetric capabilities of the cell therapy market globally. Initiation of constructive guidelines for cell therapy manufacturing and proven effectiveness of products, these are primary growth stimulants of the market.

Global Cell Therapy Technologies Market: Drivers and Restraints

The growth of cell therapy technologies market is highly driven by, increasing demand for clinical trials on oncology-oriented cell-based therapy, demand for advanced cell therapy instruments is increasing, owing to its affordability and sustainability, government and private organization , investing more funds in cell-based research therapy for life-style diseases such as diabetes, decrease in prices of stem cell therapies are leading to increased tendency of buyers towards cell therapy, existing companies are collaborating with research institute in order to best fit into regulatory model for cell therapies.Moreover, Healthcare practitioners uses stem cells obtained from bone marrow or blood for treatment of patients with cancer, blood disorders, and immune-related disorders and Development in cell banking facilities and resultant expansion of production, storage, and characterization of cells, these factors will drive the market of cell therapy technologies during forecast period.

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On the other hand, the high cost of cell-based research and some ethical issue & legally controversial, are expected to hamper market growth of Cell Therapy Technologies during the forecast period

AJune 2016, there were around 351 companies across the U.S. that were engaged in advertising unauthorized stem cell treatments at their clinics. Such clinics boosted the revenue in this market.in August 2017, the U.S. FDA announced increased enforcement of regulations and oversight of clinics involved in practicing unapproved stem cell therapies. This might hamper the revenue generation during the forecast period; nevertheless, it will allow safe and effective use of stem cell therapies.

Global Cell Therapy Technologies Market: Segmentation Analysis

On the basis of product, the consumables segment had largest market share in 2018 and is expected to drive the cell therapy instruments market during forecast period at XX % CAGR owing to the huge demand for consumables in cell-based experiments and cancer research and increasing number of new product launches and consumables are essential for every step of cell processing. This is further expected to drive their adoption in the market. These factors will boost the market of Cell Therapy Technologies Market in upcoming years.

On the basis of process, the cell processing had largest market share in 2018 and is expected to grow at the highest CAGR during the forecast period owing to in cell processing stage,a use of cell therapy instruments and media at highest rate, mainly in culture media processing. This is a major factor will drive the market share during forecast period.

Global Cell Therapy Technologies Market: Regional Analysis

North America to held largest market share of the cell therapy technologies in 2018 and expected to grow at highest CAGR during forecast period owing to increasing R&D programs in the pharmaceutical and biotechnology industries. North America followed by Europe, Asia Pacific and Rest of the world (Row).Scope of Global Cell Therapy Technologies Market

Global Cell Therapy Technologies Market, by Product

Consumables Equipment Systems & SoftwareGlobal Cell Therapy Technologies Market, by Cell Type

Human Cells Animal CellsGlobal Cell Therapy Technologies Market, by Process Stages

Cell Processing Cell Preservation, Distribution, and Handling Process Monitoring and Quality ControlGlobal Cell Therapy Technologies Market, by End Users

Life Science Research Companies Research InstitutesGlobal Cell Therapy Technologies Market, by Region

North America Europe Asia Pacific Middle East & Africa South AmericaKey players operating in the Global Cell Therapy Technologies Market

Beckman Coulter, Inc. Becton Dickinson and Company GE Healthcare Lonza Merck KGaA MiltenyiBiotec STEMCELL Technologies, Inc. Terumo BCT, Inc. Thermo Fisher Scientific, Inc. Sartorius AG

MAJOR TOC OF THE REPORT

Chapter One: Cell Therapy Technologies Market Overview

Chapter Two: Manufacturers Profiles

Chapter Three: Global Cell Therapy Technologies Market Competition, by Players

Chapter Four: Global Cell Therapy Technologies Market Size by Regions

Chapter Five: North America Cell Therapy Technologies Revenue by Countries

Chapter Six: Europe Cell Therapy Technologies Revenue by Countries

Chapter Seven: Asia-Pacific Cell Therapy Technologies Revenue by Countries

Chapter Eight: South America Cell Therapy Technologies Revenue by Countries

Chapter Nine: Middle East and Africa Revenue Cell Therapy Technologies by Countries

Chapter Ten: Global Cell Therapy Technologies Market Segment by Type

Chapter Eleven: Global Cell Therapy Technologies Market Segment by Application

Chapter Twelve: Global Cell Therapy Technologies Market Size Forecast (2019-2026)

Browse Full Report with Facts and Figures of Cell Therapy Technologies Market Report at:https://www.maximizemarketresearch.com/market-report/global-cell-therapy-technologies-market/31531/

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Global Cell Therapy Technologies Market : Industry Analysis and Forecast (2019-2026) - Publicist360

CytoDyn Files a Clinical Trial Protocol with the FDA to Treat Severely Ill COVID-19 Patients with Leronlimab where the Primary Endpoint is Mortality…

Recent data from China indicates mortality rate among COVID-19 patients requiring mechanical ventilator is more than 85%

VANCOUVER, Washington, April 01, 2020 (GLOBE NEWSWIRE) -- CytoDyn Inc. (CYDY), (CytoDyn or the Company"), a late-stage biotechnology company developing leronlimab (PRO 140), a CCR5 antagonist with the potential for multiple therapeutic indications, announced today that it has filed a second clinical trial protocol with the U.S. Food and Drug Administration (FDA) to treat severely ill COVID-19 patients with leronlimab. This trial will be conducted under the same FDA-approved IND as the Companys recently initiated Phase 2 clinical trial to treat COVID-19 patients with mild-to-moderate indications.

The Companys investigational new drug, leronlimab, has been administered to 10 severely ill patients with COVID-19 at a leading medical center in the New York City area under an emergency IND recently granted by the FDA.

CytoDyn expects to enroll patients in both the Mild-to-Moderately Ill and Severely Ill protocols very quickly under the same IND that was provided with a safe to proceed letter from the FDA. The new protocol for Severely Ill COVID-19 patients is for 342 patients, double blinded with 2:1 ratio. Patients enrolled in this trial are expected to be administered leronlimab for two weeks with the primary endpoint being the mortality rate at 14 days.

Nader Pourhassan, Ph.D., President and Chief Executive Officer of CytoDyn said, Once again, the FDA continues to be very supportive of everyones efforts to increase access to leronlimab in order to assess its therapeutic benefits for a broader range of COVID-19 patients. With a study in China indicating the mortality rate among COVID-19 patients requiring mechanical ventilator at more than 85%, the world desperately needs a therapy that can help this patient population.

About Coronavirus Disease 2019SARS-CoV-2 was identified as the cause of an outbreak of respiratory illness first detected in Wuhan, China. The origin of SARS-CoV-2 causing the COVID-19 disease is uncertain, and the virus is highly contagious. COVID-19 typically transmits person to person through respiratory droplets, commonly resulting from coughing, sneezing, and close personal contact. Coronaviruses are a large family of viruses, some causing illness in people and others that circulate among animals. For confirmed COVID-19 infections, symptoms have included fever, cough, and shortness of breath. The symptoms of COVID-19 may appear in as few as two days or as long as 14 days after exposure. Clinical manifestations in patients have ranged from non-existent to severe and fatal. At this time, there are minimal treatment options for COVID-19.

Story continues

About Leronlimab (PRO 140) The FDA has granted a Fast Track designation to CytoDyn for two potential indications of leronlimab for deadly diseases. The first as a combination therapy with HAART for HIV-infected patients and the second is for metastatic triple-negative breast cancer.Leronlimab is an investigational humanized IgG4 mAb that blocks CCR5, a cellular receptor that is important in HIV infection, tumor metastases, and other diseases, including NASH.Leronlimab has completed nine clinical trials in over 800 people, including meeting its primary endpoints in a pivotal Phase 3 trial (leronlimab in combination with standard antiretroviral therapies in HIV-infected treatment-experienced patients).

In the setting of HIV/AIDS, leronlimab is a viral-entry inhibitor; it masks CCR5, thus protecting healthy T cells from viral infection by blocking the predominant HIV (R5) subtype from entering those cells. Leronlimab has been the subject of nine clinical trials, each of which demonstrated that leronlimab could significantly reduce or control HIV viral load in humans. The leronlimab antibody appears to be a powerful antiviral agent leading to potentially fewer side effects and less frequent dosing requirements compared with daily drug therapies currently in use.

In the setting of cancer, research has shown that CCR5 may play a role in tumor invasion, metastases, and tumor microenvironment control. Increased CCR5 expression is an indicator of disease status in several cancers. Published studies have shown that blocking CCR5 can reduce tumor metastases in laboratory and animal models of aggressive breast and prostate cancer. Leronlimab reduced human breast cancer metastasis by more than 98% in a murine xenograft model. CytoDyn is, therefore, conducting aPhase 1b/2 human clinical trial in metastatic triple-negative breast cancer and was granted Fast Track designation in May 2019.

The CCR5 receptor appears to play a central role in modulating immune cell trafficking to sites of inflammation. It may be crucial in the development of acute graft-versus-host disease (GvHD) and other inflammatory conditions. Clinical studies by others further support the concept that blocking CCR5 using a chemical inhibitor can reduce the clinical impact of acute GvHD without significantly affecting the engraftment of transplanted bone marrow stem cells. CytoDyn is currently conducting a Phase 2 clinical study with leronlimab to support further the concept that the CCR5 receptor on engrafted cells is critical for the development of acute GvHD, blocking the CCR5 receptor from recognizing specific immune signaling molecules is a viable approach to mitigating acute GvHD. The FDA has granted orphan drug designation to leronlimab for the prevention of GvHD.

About CytoDynCytoDyn is a late-stage biotechnology company developing innovative treatments for multiple therapeutic indications based on leronlimab, a novel humanized monoclonal antibody targeting the CCR5 receptor. CCR5 appears to play a critical role in the ability of HIV to enter and infect healthy T-cells.The CCR5 receptor also appears to be implicated in tumor metastasis and immune-mediated illnesses, such as GvHD and NASH. CytoDyn has successfully completed a Phase 3 pivotal trial with leronlimab in combination with standard antiretroviral therapies in HIV-infected treatment-experienced patients. CytoDyn plans to seek FDA approval for leronlimab in combination therapy and plans to complete the filing of a Biologics License Application (BLA) in April of 2020 for that indication. CytoDyn is also conducting a Phase 3 investigative trial with leronlimab as a once-weekly monotherapy for HIV-infected patients. CytoDyn plans to initiate a registration-directed study of leronlimab monotherapy indication. If successful, it could support a label extension. Clinical results to date from multiple trials have shown that leronlimab can significantly reduce viral burden in people infected with HIV with no reported drug-related serious adverse events (SAEs). Moreover, a Phase 2b clinical trial demonstrated that leronlimab monotherapy can prevent viral escape in HIV-infected patients; some patients on leronlimab monotherapy have remained virally suppressed for more than five years. CytoDyn is also conducting a Phase 2 trial to evaluate leronlimab for the prevention of GvHD and a Phase 1b/2 clinical trial with leronlimab in metastatic triple-negative breast cancer. More information is atwww.cytodyn.com.

Forward-Looking StatementsThis press releasecontains certain forward-looking statements that involve risks, uncertainties and assumptions that are difficult to predict. Words and expressions reflecting optimism, satisfaction or disappointment with current prospects, as well as words such as believes, hopes, intends, estimates, expects, projects, plans, anticipates and variations thereof, or the use of future tense, identify forward-looking statements, but their absence does not mean that a statement is not forward-looking. The Companys forward-looking statements are not guarantees of performance, and actual results could vary materially from those contained in or expressed by such statements due to risks and uncertainties including: (i)the sufficiency of the Companys cash position, (ii)the Companys ability to raise additional capital to fund its operations, (iii) the Companys ability to meet its debt obligations, if any, (iv)the Companys ability to enter into partnership or licensing arrangements with third parties, (v)the Companys ability to identify patients to enroll in its clinical trials in a timely fashion, (vi)the Companys ability to achieve approval of a marketable product, (vii)the design, implementation and conduct of the Companys clinical trials, (viii)the results of the Companys clinical trials, including the possibility of unfavorable clinical trial results, (ix)the market for, and marketability of, any product that is approved, (x)the existence or development of vaccines, drugs, or other treatments that are viewed by medical professionals or patients as superior to the Companys products, (xi)regulatory initiatives, compliance with governmental regulations and the regulatory approval process, (xii)general economic and business conditions, (xiii)changes in foreign, political, and social conditions, and (xiv)various other matters, many of which are beyond the Companys control. The Company urges investors to consider specifically the various risk factors identified in its most recent Form10-K, and any risk factors or cautionary statements included in any subsequent Form10-Q or Form8-K, filed with the Securities and Exchange Commission. Except as required by law, the Company does not undertake any responsibility to update any forward-looking statements to take into account events or circumstances that occur after the date of this press release.

CYTODYN CONTACTSInvestors: Dave Gentry, CEORedChip CompaniesOffice: 1.800.RED.CHIP (733.2447)Cell: 407.491.4498dave@redchip.com

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CytoDyn Files a Clinical Trial Protocol with the FDA to Treat Severely Ill COVID-19 Patients with Leronlimab where the Primary Endpoint is Mortality...

Stem Cell Network Warns that Claims of Stem Cell Treatments for COVID-19 Are Unfounded and Misleading – GlobeNewswire

OTTAWA, March 31, 2020 (GLOBE NEWSWIRE) -- In recent weeks, a number of claims have been made that stem cells can be used as a treatment for the coronavirus disease (COVID-19). Globally, there is no clinically tested and government approved stem cell-based treatment for COVID-19.

The Stem Cell Network (SCN) urges extreme caution to those who are considering purchasing products or services advertised as a preventative or curative treatment for COVID-19. In alignment with other international stem cell and regenerative medicine organizations, SCN strongly opposes the marketing of unproven therapies and urge consumers and patients to consult with their doctor or specialist if they have questions or concerns about their health. The best way to combat the spread COVID-19 is to follow the careful advice given by Canadas Chief Public Health Officer.

Researchers across the globe are collaborating and working hard to find legitimate treatments for COVID-19, but this will take time. It is important to note that when a treatment does become available, it will be announced through recognized medical authorities, such as the World Health Organization, which is coordinating global efforts and actively compiling a database of published research on COVID-19.

For the most up-to-date information on COVID-19, please consult:World Health OrganizationPublic Health Agency of Canada

To learn more about clinical trials or stem cells:Stem Cell Network Clinical Trial FAQsCloser Look at Stem Cells

About the Stem Cell NetworkTomorrows health is here. The Stem Cell Network (SCN) is a national non-profit that supports stem cell and regenerative medicine research, training the next generation of highly qualified personnel, and delivering outreach activities across Canada. SCNs goal is to advance science from the lab to the clinic for the benefit of Canadians. SCN has been supported by the Government of Canada since inception in 2001. This strategic funding, valued at $118M has benefitted approximately 170 world-class research groups and 3,000 trainees and has catalyzed 23 clinical trials. stemcellnetwork.ca

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Stem Cell Network Warns that Claims of Stem Cell Treatments for COVID-19 Are Unfounded and Misleading - GlobeNewswire

Stem Cells, Nerves Found to Interact in Cancer Progression – Pharmacy Times

Researchers at the Institute of Oral Biology of the University of Zurich have released 2 studies that examine how stem cells promote neuronal growth in tissue regeneration and in cancer progression.

Their findings demonstrate that different stem cell populations are innervated in distinct ways and that innervation may be crucial for proper tissue regeneration, according to the study. They also demonstrate that cancer steam cells likewise establish contacts with nerves.

Stem cells can generate a variety of specific tissues that are increasingly being used for clinical application, such as the replacement of bone or cartilage, but are present in cancerous tissues and are involved in cancer progression and metastasis. Nerves are therefore fundamental for regulating the physiological and regenerative processes involving stem cells.

Using organ-on-a-chip technology, which relies on small 3-dimensional devices mimicking the basic function of human organs and tissues, the researchers demonstrated that both types of stem cells promoted neuronal growth. The dental pulp stem cells, however, yielded better results compared with bone marrow stem cells. They induced more elongated neurons, formed dense neuronal networks, and established close contacts with nerves.

Dental stem cells produce specific molecules that are fundamental for the growth and attraction of neurons. Therefore, stem cells are abundantly innervated, according to the study authors. The formation of such extended networks and the establishment of numerous contacts suggest that dental stem cells create functional connections with nerves of the face. Therefore, these cells could represent an attractive choice for the regeneration of functional, properly innervated facial tissue.

In the second study, the researchers examined the interaction between nerves and cancer stem cells found in ameloblastoma, an aggressive tumor of the mouth. They first demonstrated that ameloblastomas have stem cell properties and are innervated by facial neurons. When ameloblastoma cells were isolated and placed in the organ-on-a-chip devices, they retained not only their stem cell properties, but also attracted nerves and established contact with them.

Nerves appear to be fundamental for the survival and function of cancer stem cells. These results create new possibilities for cancer treatment using drugs that modify the communication between neurons and cancer stem cells. The researchers hope this opens unforeseen paths toward effective therapies against cancer.

The combination of advanced molecular and imaging tools and organ-on-a-chip technology offers an opportunity to reveal the hidden functions of neurons and their interactions with various stem cell types, in both healthy and pathological conditions.

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Stem Cells, Nerves Found to Interact in Cancer Progression - Pharmacy Times