Category Archives: Stem Cell Treatment

Notoginsenoside R1 promotes Lgr5+ stem cell and epithelium renovation in colitis mice via activating Wnt/-Catenin … – Nature.com

Chemicals and reagents

Notoginsenoside R1 (NGR1, BP1010, C47H80O18, purity 98%, CAS No 80418-24-2, MW: 933.13Da) was purchased from Chengdu Purifa Technology Development Co. Ltd (Chengdu, China). Dextran sulfate sodium salt (DSS, 0216011010, MW: 36kDa50kDa) was purchased from MP Biomedicals (Shanghai, China). Salicylazosulfapyridine (SASP, S0883, C18H14N4O5S, CAS No 599-79-1, MW: 398.39Da) and FITC-dextran (FD4, CAS No 60842-46-8) was purchased from Sigma-Aldrich (Darmstadt, Germany). ICG-001 (T6113, C33H32N4O4, purity 98%, CAS No 780757-88-2, MW: 548.64Da) was acquired from TOPSCIENCE (Shanghai, China). Water-DEPC treated (693520) and DMSO (D8418) were obtained from MilliporeSigma (Burlington, MA, USA).

NCM460 human intestinal epithelial cells and CT26 murine colon carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). NCM460 and CT26 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 culture medium (11875085, Gibco, NY, USA) supplemented with 10% fetal bovine serum (10099158, Gibco, NY, USA). The culture conditions included a humidified atmosphere containing 5% CO2, with a constant temperature maintained at 37C.

The Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine provided female C57BL/6 mice weighing 202g. These mice were housed in a specific pathogen-free facility under meticulously controlled conditions, including a temperature range of 2325C, humidity maintained at 60%70%, and a well-regulated 12-h light-dark cycle. The Animal Experimentation Ethics Committee of Shanghai University of Traditional Chinese Medicine granted approval (PZSHUTCM2307310004) for experimental procedures conducted on the animals. All experiments were conducted in accordance with institutional animal care guidelines and protocols approved by the committee.

According to the method reported by Yue [26], we established the acute colitis mouse model. Briefly, female C57BL/6 mice were divided randomly into four groups: Control, DSS, DSS+SASP, and DSS+NGR1. Acute colitis was induced by administering 3% DSS in the drinking water of mice for a period of 8 days. Mice in the DSS+SASP group were treated orally with SASP (260mg/kg) once per day for the same duration. The DSS+NGR1 group received NGR1 (25, 50, 125mg/kg) by oral gavage once per day for 10 days. Mice in the Control and DSS groups were administered the same volume of Control. Daily monitoring of body weight and rectal bleeding was conducted throughout the 10-day period. At the end of the experiment, mice were euthanized, and the colon was collected for further analysis.

Female C57BL/6 mice were randomly divided into four groups: DSS, DSS+ICG-001, DSS+NGR1 and DSS+ICG-001+NGR1. To establish an acute enteritis model, mice were subjected to the protocol described above. Mice in the DSS+NGR1 and DSS+ICG-001+NGR1 group were given NGR1 (25mg/kg) orally once daily for 10 consecutive days. Meanwhile, mice in the DSS+ICG-001 and DSS+ICG-001+NGR1 groups were given ICG-001 (20mg/kg) via intraperitoneal injection three times per week. The DSS and DSS+NGR1 groups received the same volume of Control.

Male BALB/c mice were acclimated for 1 week in a specific pathogen-free environment. Subsequently, CT26 cells (2105 cells/mouse) were subcutaneously transplanted into the left axillary region of each mouse. Once the tumor size reached 200mm3, the mice were randomly assigned to the vehicle group or the NGR1 group based on tumor size. Throughout the 18-day experiment, mice in the vehicle group received 0.5% CMC-Na, while those in the NGR1 group were administered 25mg/kg NGR1. Tumor volume=0.5length (mm)width (mm)2.

C57BL/6 mice were fasted for 4h before execution. Mice were then orally administered 60mg/100g body weight of FITC-dextran in 200L of sterile saline. After 4h, blood samples were collected via retro-orbital bleeding, and serum was separated by centrifugation. The serum FITC-dextran levels were measured at an excitation wavelength of 485nm and an emission wavelength of 528nm using a fluorometer (VARIOSKAN FLASH, Thermo Fisher, MA, USA).

Colonic tissues were collected from mice and fixed in 4% paraformaldehyde. Tissues were then dehydrated, embedded in paraffin, and sectioned into 4m thick slices. The sections were then stained with hematoxylin and eosin (H&E) using standard protocols. Stained sections were analyzed under a light microscope (BX61VS, Olympus, Tokyo, Japan), and images were captured for further analysis.

The concentrations of DAO (CSB-E10090m) and LPS (CSB-E13066m) in mouse serum samples were determined using the respective ELISA kit (Wuhan Huamei Biological Engineering Co., Ltd, Wuhan, China). Specifically, serum samples were added to a 96-well plate coated with DAO or LPS-specific antibodies, followed by incubation with detection reagents and substrate solution. Absorbance was measured at 450nm, and concentrations were calculated using standard curves.

Colonic tissues were fixed in 4% paraformaldehyde, embedded in OCT compound, and sectioned into 5-m slices. After permeabilization and blocking, sections were incubated with primary antibodies against ZO-1 (#13663, Cell Signaling Technology, CST, MA, USA) and Occludin (#91131, CST, MA, USA), followed by secondary antibodies conjugated to fluorophores (9300039001, ABclonal, Wuhan, China). Nuclei were counterstained with DAPI (#4083, CST, MA, USA), and images were obtained using a fluorescence microscope (BX61VS, Olympus, Tokyo, Japan). Quantification of ZO-1 and Occludin expression was performed using ImageJ software (NIH, Bethesda, MD, USA).

Colonic tissue samples were obtained from mice, fixed, dehydrated, embedded in paraffin blocks, sectioned, and stained with Alcian blue using a commercial kit. Under a light microscope (BX61VS, Olympus, Tokyo, Japan), the stained sections were examined and images were captured for subsequent analysis.

RNA was extracted using the TRIzol method, and RNA quantity and purity were measured by NanoDrop spectrophotometer (Thermo Fisher Scientific). The RNA was then reverse-transcribed using an Evo M-MLV RT Premix for qPCR kit (AG11706, Accurate Biotechnology Co., Ltd., Chengdu, China), and qPCR was performed using a SYBR Green Premix Pro Taq HS qPCR Kit (AG11718, Accurate Biotechnology Co., Ltd., Chengdu, China) (Table1). The amplification was carried out using an ABI Prism 7900HT Sequence Detection System (Life Technologies, CA, USA), and data were analyzed using the 2Ct method.

Colonic tissues were extracted and homogenized, and protein was obtained using RIPA lysis buffer with phosphatase and protease inhibitors. Protein concentration was measured using a BCA assay kit (20201ES76, Yeasen Biotech Co., Ltd, Shanghai, China). Equal amounts of protein were loaded onto SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels and separated by electrophoresis. Subsequently, the separated proteins were transferred onto PVDF membranes (000025736, Milipore, MA, USA). The membrane was then blocked with 5% BSA solution for 2h. After blocking, the membrane was incubated with primary antibodies overnight at 4C, followed by incubation with HRP-conjugated secondary antibodies for 1h at room temperature. Protein bands were visualized using ECL reagents (WBKLS0500, Millipore) and imaged with a GS-700 imaging densitometer (Bio-Rad, CA, USA). Protein expression levels were quantified using ImageJ software (NIH, Bethesda, MD, USA). The following primary antibodies were used: rabbit anti--Catenin (1:1000, #8480, CST, MA, USA), rabbit anti-p-GSK-3 (1:1000, #5558, CST, MA, USA), rabbit anti-GSK-3 (1:1000, #12456, CST, MA, USA), rabbit anti-Cyclin D1 (1:1000, #2922, CST, MA, USA), rabbit anti-c-Myc (1:1000, #5605, CST, MA, USA) and rabbit anti--actin (1:1000, #4970, CST, MA, USA).

Colonic tissue sections were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5m thick sections. Antigen retrieval was performed using citrate buffer solution (pH=6.0) and heating in a microwave oven. Non-specific binding was blocked with 5% goat serum for 30min. Sections were incubated overnight at 4C with primary antibodies, followed by incubation with a secondary antibody and staining with DAB (3,3-diaminobenzidine). Hematoxylin was used for counterstaining before the sections were examined microscopically and images were captured.

Total RNA was extracted from mouse intestinal tissues using TRIzol reagent according to the manufacturers instructions. The extracted RNA was evaluated for quality using a NanoDrop spectrophotometer (Thermo Fisher). RNA sequencing libraries were then constructed with the NEBNext Ultra RNA Library Prep Kit for Illumina, and sequencing was performed on an Illumina HiSeq platform. The differential gene was carried out on the cloud platform of majorbio (https://www.majorbio.com/).

Caco-2 cells were seeded in Millicell inserts of 24-well plates at a density of 5104 cells/400L per well. The outer chamber was filled with 600L DMEM medium (2323012, Gibco, NY, USA) and replaced every other day. TEER values were measured using a MERS00002 volt-ohm meter system (Milipore), and the electrode was sterilized with 70% ethanol and rinsed with sterile phosphate-buffered saline (PBS) before each measurement. Monolayer formation was assumed at TEER values of 400/cm2. Measurements were taken at regular intervals using the same electrode and recorded.

The intestinal crypts were isolated from the small intestine of C57BL/6 mice (6- to 8-week-old). The small intestine was removed and flushed with ice-cold PBS. The intestine was opened longitudinally and cut into 2- to 3-mm pieces. The pieces were then washed with ice-cold PBS and incubated in 3mM EDTA solution at 4C for 20min with gentle shaking. After incubation, the crypts were released by vigorously shaking the tubes. The supernatant containing the crypts was collected and filtered through a 70-m cell strainer. The crypts were then centrifuged at 1200r/min for 5min and resuspended in Matrigel (Corning, NA, USA). The crypt-Matrigel mixture was plated in 24-well plates and incubated at 37C for 30min to allow the Matrigel to solidify. The IntestCultTM OGM Mouse Basal Medium (#06005, STEMCELL, Vancouver, Canada) was then added to the wells and changed every other day.

After cultured 2 days in a 24 well plate, the intestinal crypts were randomly divided into control, DSS model group and DSS+NGR1 group. Then, the organoids were administered DSS (20g/mL), DSS (20g/mL) plus NGR1 (100M) for 4 days. The organoid growth conditions were recorded by the microscope (Olympus CKX4, Tokyo, Japan). IHC assay was conducted to examine the fluorescent protein expression of Lgr5 and -Catenin (refer to the above method of IHC).

The molecular docking was performed using AutoDock Vina software. The 3D crystal structure of -Catenin protein (PDB: 1JDH) was obtained from the Protein Data Bank (PDB) database. The structure of NGR1 was drawn and optimized using ChemDraw software and converted to a PDB file using Open Babel software. The protein and ligand files were prepared using AutoDock Tools. Docking simulations were performed and the conformation with the lowest binding energy was selected as the final docking result. The docking results were analyzed using PyMOL software.

The TOPFlash assay was performed as previously described with slight modifications [27]. HEK293T cells were seeded in 24-well plates and cultured overnight. The cells were transfected with the 500ng TOPFlash luciferase reporter plasmids (Beyotime Biotechnology, Shanghai, China) and 50ng Renilla luciferase (Promega GmbH, Mannheim, Germany) using Lipofectamine 3000 (Thermo Fisher). After 24h, the cells were treated with NGR1 (50M) and BIO (0.5M) for 24h, separately. Subsequently, cells were lysed in 150L/well passive phenylbenzothiazole (PPBT) buffer, and the luciferase activity was measured using a Dual-LuciferaseTM Reporter Assay System (Promega Corporation, WI, USA). The firefly luciferase activity was normalized to Renilla luciferase activity.

A scratch wound was created using a plastic pipette (10L) tip. NCM460 cells were then washed with PBS to remove any debris and treated with either DSS (20g/mL) or DSS (20g/mL)+NGR1 (100M) for 24h. The width of the scratch was measured using microscopy at 0 and 24h post-dosing, and the percentage of wound closure was calculated by comparing the scratch width at 24h to the initial scratch width.

NCM460 cells were treated with either DSS (20g/mL) or DSS (20g/mL)+NGR1 (100M) for 24h. Then, NCM460 cells were harvested and washed with PBS after experimental treatment. Cells were then suspended in a binding buffer containing Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and incubated in the dark at room temperature for 15min. Flow cytometry analysis was performed to detect apoptotic cells. The data were analyzed using Guava software, and the percentage of apoptotic cells was expressed.

Statistical analysis was performed using GraphPad Prism 9.0 software. Data were presented as meanstandard deviation (SD). Differences between groups were analyzed using one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant. All experiments were repeated at least three times.

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Notoginsenoside R1 promotes Lgr5+ stem cell and epithelium renovation in colitis mice via activating Wnt/-Catenin ... - Nature.com

BioCardia and StemCardia Announce Biotherapeutic Delivery Partnership – Diagnostic and Interventional Cardiology

March 15, 2024 BioCardia, Inc., a biotechnology company focused on advancing late-stage cell therapy interventions for cardiovascular disorders, andStemCardia, Inc., a biotechnology company focused on cell and gene therapy to re-muscularize the failing heart, today announced a long-term partnership to advance StemCardias investigational pluripotent stem cell product candidate for the treatment of heart failure.

Under the partnership, BioCardia is the exclusive biotherapeutic delivery partner for StemCardias cell therapy candidate through studies expected to result in FDA approval of an investigational new drug application (IND) and the anticipated Phase I/II clinical development to follow.

BioCardia has established safe and minimally invasive delivery of cellular medicines directly into the heart, said Chuck Murry, MD, PhD, StemCardias Founder and CEO. Having worked with BioCardia to successfully deliver our bona fide cardiac muscle cells in large animal models of heart failure, we are excited for this partnership to accelerate clinical development and broaden future commercial access to an off-the-shelf heart regeneration treatment.

StemCardias team encompasses recognized leaders in the field of cardiac regenerative medicine who are pursuing an elegant strategy to repair the failing heart. We look forward to supporting their efforts with our experienced team and proven, proprietary Helixbiotherapeutic delivery system, said BioCardia CEO Peter Altman, PhD. This partnership is expected to enhance future treatment options for millions of people suffering from heart failure, offset the costs of biotherapeutic delivery development for our own programs, and provide our investors with meaningful revenue sharing should our efforts together contribute to StemCardias successful therapeutic development.

For more information:www.biocardia.com

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BioCardia and StemCardia Announce Biotherapeutic Delivery Partnership - Diagnostic and Interventional Cardiology

Lawsuit over League City stem-cell treatment headed for trial – Galveston County Daily News

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Lawsuit over League City stem-cell treatment headed for trial - Galveston County Daily News

Developing Stem Cell Therapy to Halt Critical Limb Amputations – Mirage News

Critical limb ischemia is a condition in which the main blood vessels supplying blood to the legs are blocked, causing blood flow to gradually decrease as atherosclerosis progresses in the peripheral arteries. It is a severe form of peripheral artery disease that causes progressive closure of arteries in the lower extremity, leading to the necrosis of the leg tissue and eventual amputation. Current treatments include angioplasty procedures such as stent implantation and anti-thrombotic drugs, but there is a risk of blood vessel damage and recurrence of blood clots, which is why there is a strong interest in developing a treatment using stem cells.

A research team led by Dr. Sangheon Kim of the Center for Biomaterials Research at the Korea Institute of Science and Technology (KIST) announced that they have developed a three-dimensional stem cell therapy to treat critical limb ischemia through a self-assembling platform technology using a new material microgel. By using collagen microgels, a new biocompatible material, the researchers were able to easily transplant stem cells into the body and increase cell survival rate compared to 3D stem cell therapies made of cells alone.

Stem cell therapies have high tissue regeneration capabilities, but when stem cells are transplanted alone, hypoxia at the site of injury, immune responses, and other factors can reduce cell viability and prevent the desired therapeutic effect. Therefore, it is necessary to develop a material that delivers stem cells using biodegradable polymers or components of extracellular matrix as a support to increase cell viability.

The team processed collagen hydrogels to micro-scale to create porous, three-dimensional scaffolds that are easy to inject in the body and have a uniform cell distribution. Collagen, a component of the extracellular matrix, has excellent biocompatibility and cellular activity, which can induce cell self-assembly by promoting interactions between the microgel particles and collagen receptors on stem cells. In addition, the spacing between microgel particles increased the porosity of the three-dimensional constructs, improving delivery efficiency and cell survival.

The microgel-cell constructs developed by the researchers expressed more pro-angiogenic factors and exhibited higher angiogenic potential than cell-only constructs. When microgel-cell constructs were injected into the muscle tissue of mice with critical limb ischemia, blood perfusion rate increased by about 40% and limb salvage ratio increased by 60% compared to the cell-only constructs, confirming their effectiveness in increasing blood flow and preventing necrosis in the ischemic limb.

The new stem cell therapy is expected to provide a new alternative for patients with critical limb ischemia who have limited treatment options other than amputation due to its excellent angiogenic effect. Furthermore, since angiogenesis is an essential component of various tissue regeneration processes, it can be extended to other diseases with similar mechanisms to peripheral arterial disease.

"The collagen microgel developed in this study is a new biomaterial with excellent biocompatibility and high potential for clinical applications," said Dr. Sangheon Kim of KIST. "We plan to develop technologies for administration methods required in the medical field, as well as conduct follow-up research to clarify the clear mechanism of action of the treatment and discover target factors."

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Developing Stem Cell Therapy to Halt Critical Limb Amputations - Mirage News

Cell therapy for retinal degenerative disorders: a systematic review and three-level meta-analysis – Journal of … – Journal of Translational…

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Trends in Stem Cell Transplantation Refusal for Myeloma Treatment – Targeted Oncology

Determining the right path for multiple myeloma treatment is often complex as various decisions can significantly impact patient outcomes. Among these decisions, the consideration of autologous hematopoietic stem cell transplantation (HSCT) stands as a cornerstone, offering hope for improved progression-free and overall survival. However, recent research from Chakra Chaulagain, MD, showed that a small yet significant percentage of patients are refusing this potentially life-saving procedure.

An analysis of National Cancer Database (NCDB) data presented at the 2023 American Society of Hematology Annual Meeting showed that of 43,653 patients with newly diagnosed multiple myeloma recommended for HSCT, 98.05% proceeded with the procedure. However, the remaining 2% opted out. Some of the key factors influencing the patient's decision regarding HSCT related to socioeconomic, racial, and geographic disparities.

According to Chaulagain, director of the multiple myeloma and amyloidosis program at Cleveland Clinic Florida, older patients with multiple myeloma, those with comorbidities, and those lacking robust insurance coverage are more likely to decline HSCT. Furthermore, Black patients exhibited higher rates of refusal compared with White patients (OR, 1.38; P =.0022).

These findings underscore the need for future studies and policy changes to address socioeconomic and racial disparities in access to transplantation.

In an interview with Targeted OncologyTM, Chaulagain discussed the trends of rates of autologous HSCT refusal among patients with multiple myeloma.

Targeted Oncology: What led to your research on autologous HSCT refusal rates among patients with multiple myeloma?

Chaulagain: There is minimal data on real-world findings about refusal of a standard-of-care, for example, stem cell transplantation in [patients with] multiple myeloma. We wanted to explore some ideas about what are the factors that are contributing to the refusal transplant, which is the current standard-of-care, and it is known to improve both progression-free and overall survival based on randomized clinical trials. But there is limited real-world data around this subject, so we decided to investigate the NCDB.

Tumor microenvironment background with cancer cells, T-Cells, nanoparticles, molecules, and blood vessels. Oncology research concept: ratatosk - stock.adobe.com

What were the methods and design of this analysis?

This is a retrospective analysis of a very large number of [patients with] multiple myeloma that were treated by a commission of cancer-accredited cancer centers throughout the United States. There are at least 1500 of these types of cancer centers, and they report to this NCDB, where they have all of this data collected. NCDB captures about 70% of all cancer cases in the United States. We decided to get those data and analyze them just for multiple myeloma with the purpose of finding what are the variables and clinical factors that are responsible for refusal of autologous stem cell transplantation in [patients with] myeloma.

What were the key findings regarding the utilization of autologous HSCT in patients with multiple myeloma?

We had 43,600 patients [with] newly diagnosed multiple myeloma, and they were recommended to undergo a stem cell transplantation after completing their initial induction therapy by their doctors. Ninety-eight percent of the patients did go and do the stem cell transplantation, but 2% refused. We analyzed the various socioeconomic, racial, ethnic, and geographic factors about what made them refuse the stem cell transplantation.

Did the study identify any patient subgroups who were more likely to refuse?

We did find that older patients had a higher odds of refusing essential transplantation. Male [patients] had higher odds of accepting transplantation and females had higher odds of refusing it. Patients with more major medical comorbidities had higher odds of refusing it. Patients without insurance, or Medicare and Medicaid, had higher odds of refusing stem cell transplantation compared with patients who had private insurance. Median household income was also a significant predictor of whether the patient will go for a stem cell transplant or not. Those who were earning less than $63,000 annually had a higher odds of refusing autologous stem cell transplantation. Black patients, for example, had a higher odds of refusing transplantation, and Hispanic [patients] had a lower odds of refusing transplantation.

Were there any significant trends in the refusal rates over this time period?

The study time point was from 2004 until 2020. Patients who were diagnosed and treated closer to 2020 had a higher odds of refusing transplantation, and patients who were diagnosed closer to 2004 had a higher odds of accepting transplantation or lower odds of refusing transplantation, and we think it may have to do with advancement in novel therapies, particularly monoclonal antibody therapies in multiple myeloma in the current years.

What are the potential reasons as to why patients refused more than others?

The higher age, decreased income, not having strong private insurance, and also, the facility type did matter. For example, patients who were treated at nonacademic facilities had a higher odds of refusing transplant compared with patients that were treated at academic centers. There was also regional variation on whether the patient would refuse or accept transplant. For example, in South Atlantic states in the United States, patients had higher odds of refusing transplantation.

What are the implications of these findings?

We found that there was significant variation across the United States in terms of racial, economic, and geographic variation, and this data can and should be used for designing future clinical studies in a prospective basis.

How have recent advancements in the multiple myeloma space such as the emergence of novel therapies impacted transplantation?

Based on our studies, the emergence of novel therapies and immunotherapy, particularly anti-CD38 monoclonal antibodies like daratumumab [Darzalex], have led to decrease utilization of transplant, and it will probably further evolve down the road because of the availability of even more effective novel therapies such as [chimeric antigen receptor] T-cell therapy, and bispecific T-cell engager therapy. The role of transplant will continue to evolve and will probably continue to diminish down the road.

What barriers still need to be addressed regarding transplant?

These are bigger decisions at the policy and procedure and legislation [levels], like increasing incidence coverage, increasing socioeconomic aspects for all of our patients, particularly those who are marginalized or who are minorities. This is a bigger, national goal and the legislator has to act on it,

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Trends in Stem Cell Transplantation Refusal for Myeloma Treatment - Targeted Oncology

Japanese hospital to evaluate technology used in European trials – Labmate Online

Leading stem cell researchers at Shonan Kamakura General Hospital (SKGH), Japan, are collaborating with regenerative cell therapy developer CellProthera to manufacture autologous endothelial progenitor cells (EPCs) for use in forthcoming clinical trials. Led by world-renowned stem cell expert Takayuki Asahara, MD, PhD, the SKGH research team will use the companys automated manufacturing technology, along with single-use cell culture kits to produce therapies for patients with ischemic and renal diseases.

Professor Asahara, Deputy Director of Shonan Research Institute of Innovative Medicine atSKGH, was the first researcher to isolate EPCs from peripheral blood. EPCs are naturally deployed in the body to repair blood flow after it is restricted (as in ischemic stroke).

CellProtheras StemXpand, which has been in use in European trials to grow patients own cells into a therapeutic dose, will be rigorously tested to meet SKGHs manufacturing specifications and adapted as needed to begin qualification runs for an upcoming clinical trial. After the collaborators confirm consistency and reproducibility both in the manufacturing process and with the previously manufactured product, Prof. Asaharas team will perform validation runs to ready the technologys use for clinical testing.

We are honoured to work with Prof. Asahara given his ground-breaking experience in the regenerative medicine space and think he is the ideal partner to demonstrate the utility of our manufacturing technology beyond our own pipeline, said Matthieu de Kalbermatten, CEO, CellProthera. As a long-time advocate for the use of stem cells for the treatment of ischemic and renal diseases, I am hopeful this collaboration will pave the way for the StemXpand and StemPack to play a pivotal role in the research and development of stem cell treatments across the globe.

Ischemic diseases remain one of the leading causes of death in Japan, with limited treatment options, commented Prof. Asahara. We hand-picked CellProthera for collaboration based in part on how StemXpand, a tried and trusted technology, will help us meet the needs of patients with ischemic diseases through our development of targeted stem cell therapies.

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Stem cell therapy for MS seen to lower mitochondrial DNA in study – Multiple Sclerosis News Today

People with multiple sclerosis (MS) have higher amounts of mitochondrial DNA in their spinal fluid, which surrounds the brain and spinal cord, than do their healthy counterparts, a small study found.

Mitochondria are small cellular organelles that produce most of the energy needed to power cells. These cell powerhouses have their own DNA, which can be released into the surrounding fluid and contribute to inflammatory processes.

In this study, however, researchers also observed that treatment with stem cell therapy brought mitochondrial DNA to near-normal levels, suggesting it could be a marker of disease activity and response to treatment for MS.

The study, Cerebrospinal fluid mtDNA concentrations are increased in multiple sclerosis and were normalized after intervention with autologous hematopoietic stem cell transplantation, was published in Multiple Sclerosis and Related Disorders.

MS occurs when healthy parts of the brain and spinal cord become inflamed and damaged, causing a range of symptoms. It is thought that certain immune cells in MS may go haywire and burst open, releasing DNA from their mitochondria that boosts further inflammatory processes.

Earlier work has suggested that changes in mitochondria occur early in the disease course and contribute to neurodegeneration. However, treatment with certain disease-modifying therapies seems to reduce the amount of mitochondrial DNA known as mtDNA that gets released into the cerebrospinal fluid, or CSF.

Now, researchers in Sweden conducted a study involving MS patients and healthy people. There were two main goals: First, to determine if MS patients indeed have higher levels of mitochondrial DNA in the CSF than do healthy controls, and second, to investigate if treatment with a stem cell transplant could lower the levels of that DNA in patients. Such a transplant, known as autologous hematopoietic stem cell transplantation (aHSCT), uses cells collected from the patient.

The study included 48 people with relapsing-remitting MS and 32 healthy individuals. The results showed that the MS patients had significantly more copies of mtDNA than did the healthy individuals (a median of 16 vs. 5.6 copies per microliter of CSF).

The amount of mtDNA was not impacted by sex or age in either group, but correlated with the number of relapses, disability levels, and a shorter disease duration in patients. Levels of mtDNA also were linked with higher the levels of neurofilament light chain (NfL), a marker of nerve cell damage, and a higher number of lesions with active inflammation.

All patients planned to undergo a stem cell transplant, a procedure that aims to reset the immune system. It involves collecting a patients own hematopoietic stem cells immature cells that can develop into all types of blood cells then wiping out the entire immune system with a round of chemotherapy or radiation therapy, and infusing the stem cells back to give rise to new immune cells.

By generating immune cells that are not primed to attack the brain and spinal cord, the procedure is expected to reduce the inflammation and nerve cell death that drives MS.

One year after the transplant, the results showed, the median number of mtDNA copies decreased significantly, from 16 to 5.9 copies per microliter. That number remained significantly reduced in the years that followed, at 8 copies per microliter after two years, and 7.2 copies per microliter after 3-5 years.

mtDNA concentrations were normalized in MS patients after intervention with aHSCT, the researchers wrote.

After the transplant, a total of 39 patients retained a status called no evidence of disease activity (NEDA-2), meaning they experienced no new MRI activity and no relapses over the follow-up period. The other nine patients had evidence of inflammatory disease activity (EIDA), and had experienced at least one of these events.

Data showed that patients with EIDA had significantly higher levels of mtDNA one year after the transplant that those with NEDA-2 (10 vs. 5.2 copies per microliter). The difference remained significant after two years (18 vs. 7.1 copies per microliter), but not after 3-5 years.

These results position mtDNA as a potential biomarker for monitoring inflammatory activity and response to treatment in MS, the researchers wrote. In addition, our study adds to the growing evidence base for the therapeutic efficacy of aHSCT.

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Stem cell therapy for MS seen to lower mitochondrial DNA in study - Multiple Sclerosis News Today

Targeting PRMT9-mediated arginine methylation suppresses cancer stem cell maintenance and elicits cGAS-mediated … – Nature.com

Ethics statement

This study follows ethical regulations. Experiments using patient specimens were approved in part by the institutional review boards of City of Hope Comprehensive Cancer Center (COHCCC) and conducted in accordance with the Declaration of Helsinki (2013). Samples were acquired as part of the COHCCC institutional review board-approved clinical protocol no. 18067. All mouse experiments were completed in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at COHCCC. Experiments were performed in accordance with a protocol approved by the COHCCC ICUC (no. 15046). The maximum tumor size (humane endpoint) permitted by IACUC is 15mm (diameter). All animals were euthanized before tumor size reached 15mm in diameter. Maximum tumor size did not exceed 15mm.

De-identified, clinically annotated primary patient samples including those derived from peripheral blood or bone marrow were obtained from patients with AML at COHCCC. The annotations are shown in Supplementary Table 1. Normal cells derived from peripheral blood were obtained from the COHCCC. Informed written consent was completed and acquired from all involved participants before sample acquisition. MNC separation, CD34+ cell enrichment or CD3+ T cell depletion was performed as described previously58.

Molm13 (catalog no. ACC 554, DSMZ), MV4-11 (catalog no. CRL-9591, ATCC), THP1 (catalog no. TIB-202, ATCC), NB4 (catalog no. ACC 207, DSMZ), U937 (catalog no. CRL-1593.2, ATCC), HL-60 (catalog no. CCL-240, ATCC), MA9.6ITD and RAJI (catalog no. ACC 319, DSMZ), UPN1 (catalog no. CVCL_A795, Cellosaurus), BL41 (catalog no. ACC 160, DSMZ), Rec1 (catalog no. ACC 584, DSMZ), OCI-Ly3 (catalog no. ACC 761, DSMZ) and A20 (a gift from Y. Fu) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FCS as described previously58,59. All other cell lines, including 293FT (catalog no. R70007, Thermo Fisher Scientific), DMS273 (a gift from R. Salgia), DMS114 (a gift from R. Salgia), SW1573 (a gift from E. Wang), A549 (a gift from E. Wang), SW620 (catalog no. CCL-227, ATCC), HCT116 (catalog no. CCL-247, ATCC), HepG2 (catalog no. HB-8065, ATCC), PC3 (a gift from S. Priceman), DU145 (a gift from S. Priceman), MDA-MB-231 (catalog no. CRM-HTB-26, ATCC), HT1197 (catalog no. CRL-1473, ATCC), A172 (catalog no. CRL-1620, ATCC), MIAPACA2 (catalog no. CRM-CRL-1420, ATCC) and HT1080 (catalog no. CCL-121, ATCC) were cultured in DMEM with 10% FCS. MA9.6ITD cells (MLL-AF9 plus FLT3-ITD) were established by J. Mulloy60. The human primary normal and AML CD34+ cells used for transduction were maintained as described previously59. Specifically, as noted in that paper, the medium was StemSpan SFEM (STEMCELL Technologies) supplemented with 50ngml1 recombinant human stem cell factor (SCF), 100ngml1 Flt3 ligand (Flt3L), 100ngml1 thrombopoietin, 25ngml1 interleukin-3 (IL-3) and 10ngml1 IL-6 (PeproTech). Mouse AML cells were cultured in RPMI 1640 medium with cytokines (mouse IL-3, 10ngml1; mouse IL-6, 10ngml1; mouse SCF, 30ngml1; Supplementary Table 10) as described previously59.

In all experiments, male and female, 610-week-old, WT C57BL/6J (strain no. 000664, The Jackson Laboratory), B6(Cg)-Rag2tm1.1Cgn/J (strain no. 008449, Rag2/, The Jackson Laboratory), B6(Cg)-Ifnar1tm1.2Ees/J (strain no. 028288, Ifnar1/, The Jackson Laboratory), Kmt2atm2(MLLT3)Thr/KsyJ (strain no. 009079, MLL-AF9 knock-in, The Jackson Laboratory), B6.129S(C)-Batf3tm1Kmm/J (strain no. 013755, Batf3/, The Jackson Laboratory), NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (strain no. 005557, NSG, The Jackson Laboratory), NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (strain no. 013062, NSGS, The Jackson Laboratory) and NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1em1Mvw H2-D1tm1Bpe Il2rgtm1Wjl/SzJ (strain no. 025216, NSG-MHC I/II DKO, The Jackson Laboratory) mice were used. B6-Ly5.1 (CD45.1, NCI 564) and BALB/c (NCI 028) mice were available from an outside vendor. Male and female mice were housed at the COH Animal Resource Center. All care and experimental procedures followed established institutional guidelines. The mouse room is conditioned with a 14h light10h dark cycle, temperatures of 6575F and 4060% humidity. The procedure was run in accordance with a protocol approved by the IACUC at COHCCC.

Mouse experiments were performed once: Fig. 2d,e,h (male and female; five WT B6 mice per group); in Fig. 2f,g (male and female; five WT B6 mice per group); Fig. 2q (male and female; eight NSGS mice per group); Fig. 2r (male and female; eight NSGS mice for Ctrl, seven NSGS mice for Prmt9 KD); Extended Data Fig. 2p (male and female; six Prmt9loxP/loxP/Mx1Cre mice for Prmt9 WT, nine Prmt9loxP/loxP/Mx1Cre+mice for Prmt9 KD); Extended Data Fig. 2q (male and female; eight Prmt9loxP/loxP/Mx1Cre mice for Prmt9 WT, 15 mice (Prmt9loxP/loxP/Mx1Cre+) for Prmt9 KD); Extended Data Fig. 2r (male and female; seven B6-Ly5.1 mice per group); Fig. 5d (male and female; seven WT B6 mice per group); Fig. 5e (male and female; five Rag2/ mice per group); Fig. 5f(male and female; five NSGS mice per group); Fig. 5g (male and female; seven WT B6 mice per group); Fig. 5s (male and female; five WT B6 mice for naive mice, four survival mice from Fig. 5d for survivors); Fig. 5v (male and female; five Ifnar1/ mice for Ifnar1 KO, six WT B6 mice for Ifnar1 WT); Fig. 6j (male and female; seven WT B6 mice for the Prmt9 KD group, five WT B6 mice for each of the other three groups); Fig. 6m (male and female; seven WT B6 mice for cGAS KO+cGASN group, five WT B6 mice for each of the other two groups); Fig. 6s (seven WT B6 mice for each Batf3 WT group, five Batf3/ mice for the Batf3 KO group); Extended Data Fig. 7c (seven WT B6 mice for the Ctrl and Prmt9 KD groups, five WT B6 mice for the T and NK cell depletion groups); Extended Data Fig. 9gi (five BALB/c mice per group); and Extended Data Fig. 9j,k (five NSGS mice per group). scRNA-seq and bulk RNA-seq were performed once per sample and are shown in Figs. 1e, 5h and 6c. If not otherwise specified, in vitro experiments were repeated at least three times.

The CD530-EF1A-IRES-GFP vectors were purchased from System Biosciences. The CD530-EF1A-T2A-GFP vectors were modified from CD530-EF1A-IRES-GFP, replacing IRES with T2A sequences. Full-length WT or LDIG-to-AAAA mutant PRMT9 (ref. 29) were cloned into CD530-EF1A-IRES-GFP vectors. FLAG-tagged XRN2 and FLAG-tagged DDX3X variants, and FLAG-tagged either full-length WT or C-terminal (amino acids 436636) PABPC1 or R493K, R481K, R506K or 3RK mutants were cloned into the CD530-EF1A-T2A-GFP vector. All plasmids were synthesized by Genscript. shRNAs targeting human PRMT9, mouse Prmt9, PABPC1 and CREB1 were purchased from Sigma-Aldrich (MISSION shRNA) and cloned into pLKO-SFFV-RFP, as described elsewhere58. cGAS WT and the activation mutant N were purchased from Addgene and constructed into a DOX-inducible expression vector. SMARTvectors with shPRMT9 were purchased from Dharmacon (Horizon Discovery). The oligonucleotides used are listed in Supplementary Table 11.

Compounds were sourced from the NCI Developmental Therapeutics Program (DTP), ZINC libraries or MolPort. The PEGylated liposome packaging of LD2 used for animal treatment was prepared using the thin film hydration method. Lipids (distearoylphosphatidylcholine, cholesterol and DSPE-PEG(2000) at a ratio of 3:1:0.2) plus compound were dissolved in chloroform; then, organic solvent was separated in a vacuum to form a thin film. Subsequently, lipids were hydrated in PBS, pH 7.4, at 60C to form liposomes.

Virus production was as described previously61. HEK 293T cells were transfected with pMD2.G and psPAX2 packaging vectors plus lentivectors designed to overexpress or knock down genes using the calcium phosphate method as described previously61. Supernatants containing virus particles were filtered and concentrated. Viral infection was performed as described previously61.

RNA was prepared according to the TRIzol reagent protocol. After generation of complementary DNA, qPCR with reverse transcription was performed as described previously59. The primers used are listed in Supplementary Table 11.

Cell lysates were prepared in a buffer containing 50mM Tris, pH 7.4, 150mM NaCl and 1mM EDTA supplemented with protease inhibitors. Cell lysates were incubated with anti-FLAG beads or interested primary antibody (Sigma-Aldrich) overnight and denatured for immunoblotting. Proteins of interest were probed with primary and secondary antibodies. Signals were detected using the SuperSignal West Pico or Femato kits. All immunoblots were imaged using the G:BOX Chemi XX6 gel doc system and quantified with the ImageJ software (NIH).

Samples were prepared according to the protocol of the SimpleChIP Plus Enzymatic Chromatin IP Kit (catalog no. 9005, Cell Signaling Technology). Immunoprecipitates were exposed to anti-CREB1 (catalog no. SC-240, Santa Cruz Biotechnology) and anti-H3K27Ac antibodies, plus Protein G magnetic beads. After reversing, DNA was enriched; this was followed by qPCR.

Cells derived from the bone marrow or spleen samples were washed with PBS containing 1% FCS and then passed through a single-cell strainer and subjected to lysis of red cells. Before flow cytometry, cells were stained with the indicated antibodies in the same buffer. Flow cytometry analysis was performed. Data analysis was performed using FlowJo v.10. Molm13 cell engraftment in mice was determined using an anti-human CD45 antibody. CD45.2+ donor cells from transplants were determined using anti-mouse CD45.1 and CD45.2 antibodies. Mouse HSPCs were determined by staining with anti-mouse lineage antibody, including cKit, Sca-1, CD16 and CD32, and CD34 antibodies and a lineage antibody cocktail, including anti-mouse CD3, CD4, CD8, CD11b, CD11c, CD19, CD41, Ter119, B220, IgM, NK1.1, Gr-1 and interleukin-7 receptor subunit alpha (IL-7R). Anti-mouse Mac1, Gr-1, B220 and Ter119 were used to define mouse bone marrow differentiation. We also detected antigen-specific T cells in tumors as described previously44. For intracellular staining, fixed cells were incubated once with antibodies against IFN- (clone XMG1.2) and granzyme B (clone QA16A02). To define the human primary samples, we used the following markers: T cells (CD3+), B cells (CD19+/CD20+), monocytes (CD14+) and DCs (HLA-DR+CD34CD33CD3CD19CD20CD14CD56), as well as the immature CD33+CD34+CD45dim subset. CD69 and IFN- staining was used to determine T cell status. For the cell cycle studies, fixed cells were stained with 4,6-diamidino-2-phenylindole (DAPI).

Bone marrow cells (0.5106per transplant) from CD45.2+ Prmt9loxP/loxPMxCre+ or Prmt9loxP/loxPMxCre mice were combined with CD45.1+ bone marrow cells (at 1:1 ratio) and then implanted into lethally irradiated (900cGy) B6-Ly5.1 mice by intravenous injection. Peripheral blood samples were collected and assessed with CD45.1 and CD45.2 antibodies. Mouse recipients were induced with pIpC (InvivoGen) intraperitoneally 15mgkg1 every other day for 7 days; CD45.2+ chimerism in peripheral blood was assessed every 4 weeks.

For the limiting dilution assays, to evaluate LSC frequencies, AML cells were suspended in Colony Forming Cell growth medium with DOX to induce Prmt9 KD and plated in multi-well plates. To evaluate the frequency of leukemia-initiating cells in vivo, bone marrow cells isolated from Ctrl or Prmt9 KDMA9 AML mice were injected intravenously into sublethally conditioned recipient mice, as described in Supplementary Table 7. The number of recipient mice with leukemia development was determined in each group. The frequency of LSCs and LICs was determined using the ELDA software.

To assess the effect of Prmt9 KO and KD in vivo, MA9 or CMM cells were transduced with lentiviral vectors harboring a luciferase reporter. Cells were used for intravenous inoculation into sublethally irradiated CD45.1 B6 mice or WT B6, Rag2/ or NSGS mice. As for bioluminescence imaging, mice were administered 150mgkg1 d-luciferin (GoldBio) within PBS, followed by analysis using Lago X. Bioluminescent signals were quantified using the Aura imaging software (Spectral Instruments Imaging). Total values were determined using the regions of interest and photonsscm2sr. To identify the immune subsets contributing to leukemia regression after Prmt9 KD, we performed antibody-based depletion with an initial dose of combined anti-CD4 and anti-CD8 treatment or anti-NK1.1 treatment administered 1 day before in vivo DOX administration to Prmt9 KD mice. Antibodies (400g) were injected intraperitoneally twice the first week, and then at 200g twice weekly to maintain NK or T cell depletion. To assess DC function in Prmt9 KD outcomes, we implanted Batf3 WT or Batf3 KO mice with AML cells for further evaluation.

Cell growth was assessed using the CellTiter-Glo Assay Kit (Promega Corporation). Apoptosis was determined using annexin V or DAPI. Colony formation capacity was determined as described previously58,59.

For SILAC, Molm13 cells were cultured in SILAC RPMI 1640 medium (catalog no. 88365, Thermo Fisher Scientific) with 10% FCS (catalog no. A3382001, Thermo Fisher Scientific) and either light l-lysine (catalog no. 89987, Thermo Fisher Scientific) and l-arginine (catalog no. 89989, Thermo Fisher Scientific) for control cells, or heavy lysine (catalog no. 88209, Thermo Fisher Scientific) and l-arginine (catalog no.89990, Thermo Fisher Scientific) for inducible PRMT9 KD cells, for at least ten passages to ensure full incorporation of light or heavy l-lysine and l-arginine.

After 3 days of DOX induction in both control and PRMT9 KD cells, light-labeled and heavy-labeled cells were combined at 1:1 ratio. Cells were washed and centrifuged at 300g for 5min. Cell pellets were lysed in 9M urea with protease and phosphatase inhibitors in HEPES (pH 8.0) buffer. Samples underwent four cycles of sonication for 30s each using a microtip sonicator (VibraCell VCX130, Sonics & Materials) operating at 50% amplitude. Lysates were centrifuged at 20,000g for 15min; protein quantification was performed by using a bicinchoninic acid (BCA) assay. An equal amount of extracted protein from heavy and light SILAC culture was mixed for further digestion. The sample was first reduced by incubation with dithiothreitol (DTT) (5mM, 55C) and then alkylated by incubation with iodoacetamide (10mM) in the dark. The sample was diluted fourfold before sequential digestion first with LysC (2h) and then overnight with Trypsin Gold. Digestion was quenched using trifluoroacetic acid and the sample was desalted using 0.7ml of a Sep-Pak Classic C18 column (Waters). Eluted peptides were speedvacd to dryness and reconstituted in 1.4ml immunoaffinity purification buffer followed by peptide quantification using a BCA assay. We subjected 5% of peptides to global quantitative proteomics analysis and 95% of the rest to methyl-R peptide enrichment. This consisted of sequential incubation of peptides with anti-MMA antibody beads (catalog no. 12235, Cell Signaling Technology) and anti-SDMA antibody beads (catalog no. 13563, Cell Signaling Technology). Enriched peptides were reconstituted in 10l loading solvent (98% water, 2% acetonitrile, 0.1% formic acid); 1g of nonenriched peptides was used for global protein identification.

Data were obtained on an Orbitrap Fusion Lumos mass spectrometer (methylated peptides) or Orbitrap Eclipse with FAIMS Pro interface (unmodified peptides) coupled to a U3000 RSLCnano LC system with running binary solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at 300nlmin1. Methylated peptides (5l per injection) were directly loaded on a 25cm EasySpray C18 column and eluted over a 120-min gradient as follow: 80min with 219% B, 20min with 1930% B, 5min with 3098% B, followed by 2min of high organic wash and return to initial conditions in 1min. Unmodified peptides (1g peptides, 5l per injection) were directly loaded on a 50-cm EasySpray C18 column and eluted over 240min using the following gradient: 12min with 25% B, 158min with 519% B, 40min with 1930% B, 9min with 3090% B, followed by 4min of high organic wash and return to initial conditions in 2min. Using a duty cycle of 3s (Lumos) or 1s (Eclipse) per FAIMS CV (40/60/80), most abundant precursors were fragmented using higher-energy collisional dissociation (32% normalized collisional energy on Eclipse and 35% normalized collisional energy on Lumos) and measured in the ion trap. Dynamic exclusion was set to 60s to prevent resampling of previously analyzed precursors.

MS raw files were searched against the human UniProt protein database (downloaded in 2020, 42,373 entries) and a common contaminant database using MaxQuant v.1.6.17.0. The results were filtered to 1% protein and site false discovery rate (FDR). The resulting methyl peptide SILAC ratios obtained from the MaxQuant evidence.txt output file were normalized to their protein SILAC ratios before further analyses62.

Motif analysis was performed using the iceLogo web application as described previously30.

We performed polysome profiling as described previously28. Engineered Molm13 cells were DOX-induced for 3 days to delete PRMT9 expression and then treated for 5min with 100gml1 cycloheximide. After treatment, cells were collected and lysed. We prepared sucrose density gradients (1545% w/v) using a Gradient Master (BioComp Instruments). Then, the supernatant from the cell lysates was separated using centrifugation and fractionation. The collected RNA was further assessed in the qPCR analysis.

Protein synthesis was assessed by using the Click-iT Plus OPP Assay Kit (Thermo Fisher Scientific), with modifications. Briefly, treated cells were exposed to Click-iT OPP, then washed with PBS and fixed. After permeabilization for 15min, cells were reacted with cocktail, then analyzed using flow cytometry.

The assay was performed in a 30-l reaction with 50mM Tris HCl, pH 7.4, 50mM NaCl, 50mM KCl, 1mM MgCl2 and 1mM DTT buffer. Specifically, 1g purified PABPC1-CT protein or synthesized peptides, 1g purified PRMT9 protein and 5M of SAM (Cayman Chemical) were combined. Methylated proteins and peptides were detected with immunoblot or dot blot assays using anti-pan-SDMA, anti-pan-MMA, anti-pan-ADMA or our in-house PABPC1 R493me antibody. The R493me antibody was created by Genemed Synthesis. For the ex vivo tritium labeling of the methylation assay, 1g purified PRMT9 protein, 1g HA-tagged PABPC1 WT or corresponding PABPC1-R481K/R493K/R506K (3RK) protein, which were immunoprecipitated from 293T cells, and 1l S-adenosyl-l-[methyl-3H] methionine (78Cimmol1) was added to a 30l reaction mixture at 30C for 1h. Samples were separated and transferred to polyvinylidene membranes for further assessment.

The crystal structure of human PRMT9 (Protein Data Bank (PDB) ID 6PDM; 2.45A resolution) was used for virtual screening. Missing loops were added using a molecular operating environment loop modeler. A box size of 2521273 centered around the cocrystalized chemical probe was used for screening, which includes both the SAM pocket and catalytic pocket in the N-terminal methyltransferase domain (amino acids 150520). To rank the binding affinity, parallel AutoDock Vina63,64 runs were conducted on a local computer cluster. Seven hundred thousand compounds from the ZINC library were selected using the following criteria: molecular weight 350450, log P<3, total charge 2e to +2e and availability. In addition, we also screened the NCI library (NCI DTP 260,000 compounds). Each ligand was docked ten times and ranked according to the lowest binding energy score. After screening, we purchased the top 300 candidates (142 of them were available) from the NCI DTP and the top 100 candidates (70 of them were available) from the ZINC library to assess anti-AML activity. To estimate lead compound selectivity, we also performed Vina docking of LD2 into human CARM1 (PDB ID 5U4X), PRMT5 (PDB ID 4X61), PRMT7 (PDB ID 4M38) and PRMT9. To compare LD2 binding to PRMT5 versus PRMT9, we carried out two replicas of 100-ns molecular dynamics simulation of LD2 docked into each.

Maltose binding protein (MBP)-tagged PRMT9 core methyltransferase domain (150474) protein was expressed and purified by Genscript. Briefly, the PRMT9 core methyltransferase domain sequence was inserted into the pMAL-c5X vector between the Nde I and EcoR I sites. Tagged protein was expressed in BL21 and purified on an MBP column, followed by Superdex 200 and Q Sepharose columns. Proteins were sterile-filtered and lyophilized after extensive dialysis against the NMR buffer (50mM NaH2PO4, pH 7.5). Deuterium oxide-based sodium phosphate buffer was used with 5% DMSO-d5. For the STD NMR assay, the molar ratio of LD2 to PRMT9 was 60:1 in which the concentration of PRMT9 was 0.67M; 50M trimethylsilylpropanoic acid-d4 was used as the internal reference. The molar ratios between PMRT9 and LD2 were 1:20, 1:40 and 1:60, in addition to a control sample with free LD2. LD2 concentration in the CarrPurcellMeiboomGill (CPMG) experiments was 40M. The NMR saturation transfer difference (STD) experiments were carried out at 25C on a 700-MHz Bruker Ascend system equipped with a 5-mm triple resonance cryogenic probe as described previously65. The CPMG experiment was performed as described previously66. Data were analyzed using Bruker TopSpin v.3.6.

We also assessed whether LD2 binds to PRMT9 directly in vivo; to do so, a cellular thermal shift assay was performed as described previously39,40. We first engineered Molm13 cells to overexpress FLAG-tagged PRMT9 WT or PRMT9 mutant (W152A, D258A and E433A; all three residues are predicted drug and PRMT9 binding sites). Five million cells were pretreated with 2.5M LD2 overnight. DMSO was used as the control. Cells were aliquoted in each tube and heat-shocked using Thermal Cycler at the indicated temperatures. Cells were then lysed for the immunoblot assay. Experiments were performed using three biological replicates.

Two million MNCs from AML bone marrow specimens were cultured per well in 24-well plates in IMDM plus 20% FCS under physiological cytokine conditions as described previously41,42 (granulocyte-macrophage colony-stimulating factor in 200pgml1, granulocyte colony-stimulating factor in 1ngml1, SCF in 200pgml1, IL-6 in 1ngml1, macrophage inflammatory protein-1 alpha in 200pgml1 and leukemia inhibitory factor in 50pgml1). We then used the EasySep Dead Cell Removal Kit (STEMCELL Technologies) to ensure more than 95% living cells before culture. Cells were treated with vehicle (dimethylsulfoxide), 2.5M LD2, anti-PD-1 (pembrolizumab, 10gml1, SIM0010, Bio X Cell) or LD2 plus anti-PD-1 for 4 days at 37C. On day 4, cells were pretreated for 6h with brefeldin A and subjected to CyTOF immunostaining with customized surface or intracellular marker antibodies, according to Fluidigm CyTOF protocols (PN400279A4). An untreated peripheral blood mononuclear cell sample from a healthy donor served as a control for phenotyping. Samples were acquired on a Fluidigm Helios. Data were normalized and saved as FCS files before analysis using the Cytobank software (https://premium.cytobank.org/). After data were cleaned up, spanning-tree progression analysis for density-normalized events was used to cluster AML cells and immune cell subpopulations based on the median level of each.

For CD8A, CD8B, GZMA, GZMB and PRF1, the average expression levels of these genes were used to estimate CTL levels in AML samples43,67. We carried out in silico tests to calculate the ratio of PRMT9hi and PRMT9lo patients exhibiting high versus low CTL scores using both GSE144688, which includes 526 samples of patients with AML, and GSE12417, which includes 163 patient samples. For each patient, high versus low CTL scores were decided according to cutoff of 0.5 for the z-score. A Fishers exact test was used to assess significance.

Bone marrow cells in MA9-transplanted mice, and bone marrow and spleen cells in Ctrl and Prmt9 KD mice administered DOX in drinking water over 7 days, were collected for analysis. Single cells were resuspended in 0.4% BSA and loaded to generate an emulsion of single-cell gel beads. Approximately 5,00010,000 cells were loaded per channel. Libraries were prepared using the Single Cell 3 Library & Gel Bead, Single Cell 3 Chip and i7 Multiplex Kits, according to the Single Cell 3 Reagent Kits v2 User Guide (part no. CG00052 Rev A). Libraries were sequenced on an Illumina HiSeq 4000 system.

We used the Cell Ranger Single Cell Software Suite to perform single-cell 3 gene counting and aggregation of multiple samples to generate raw counts, cell barcodes and gene features. The R package Seurat was run as the platform to implement all data processing procedures68.

Cell quality control was executed as follows: the minimum detected genes (3) in each cell; the minimum number of cells (200) related to each gene; and the maximum fraction (0.2%) of counts from mitochondrial genes per cell barcode. The high-count depth threshold (2,000) was used to filter out potential doublets. Then, the count matrix was normalized to obtain the correct relative gene expression abundance between cells69. Then, the R package Harmony was applied to remove batch effects due to biological differences between cell types or states.

To retain informative genes with high variability, genes with small variations (below 2) among all cells were filtered out. Then, the dimensions of count matrices were reduced using dedicated dimension reduction algorithms, such as UMAP and t-distributed stochastic neighbor embedding (t-SNE). Two-dimensional visualization outputs were then generated using the leading reduced components in the UMAP and t-SNE plots.

UMAP-related processed data were regarded as the input of cell clustering. Neighborhood distances among all cells were determined to infer the identity of each cell. Then, clusters were acquired via specified distance metrics (Euclidean distance). Furthermore, for each cluster, the R package MAST was used to deduce significant DEGs. These DEGs were considered markers of a cluster and were used for annotation purposes. Annotations were conducted manually by comparing marker genes with the literature and arranging cell categories. In addition, automatic annotation of cell clusters was done using the R package SingleR, as described previously70. By combining both annotation styles, the final cell type labels of each cluster were acquired.

For the cell type clusters of interest, GSEA was performed based on preordered genes ranked using MAST-derived (log10(Padj)sign (log fold change)) with 1,000 permutations71. The gene sets of the Hallmark, Kyoto Encyclopedia of Genes and Genomes, chemical genetic perturbation and Gene Ontology-Biological Process categories of the Molecular Signatures Database were considered as the signatures. Finally, specific enriched genes within a cluster were visualized by averaging their expression among all cells in that cluster. Key enriched gene expression was rescaled by z-scores and visualized in the heatmap.

scRNA-seq uncovered ten distinct T cell clusters (c0c9). c0 cells expressed Cd4 and CD62L, but not the effector and memory T cell marker Cd44 or T cell activation genes. Thus, c0 was defined as naive CD4+ T cells. Similarly, c1 cells expressed Cd8a and CD62L but not Cd44 or other T cell activation markers and were defined as naive CD8+ T cells. c2 cells expressed Cd8a, Cd44 and Sell, and intermediate levels of Tbx21 (T-bet) and Eomes, and represented a memory CD8+ T cell population. c3 cells expressed high Cd4, Cd44 and Icos, Ctla4, Tnfrsf4 and Pdcd1, but did not express CD62L and were defined as activated and effector CD4+ T cells. c5 cells expressed Cd44 and showed the highest levels of Ifng, Gzmb, Icos, Tim-3, Il2ra, Tnfrsf18 and Lag3, considered as differentiated CTLs. c6 cells were defined as Treg cells because they express Cd4, Il2ra (Cd25) and Foxp3. c4, c7, c8 and c9 cells contained both CD4+ and CD8+ T cells. c4 and c9 showed lower levels of activation markers, and lower CD62L and higher Cd44, suggesting that they represent Teff cell populations. c7 expressed only the naive T cell marker CD62L, indicating a naive population, while c8 expressed lower CD62L and higher Cd44, but did not express other T cell activation markers, suggesting it represents a memory T cell population.

Total RNA was prepared using the TRIzol reagent (Thermo Fisher Scientific). RNA quality (RNA integrity number) was assessed and sequenced on an Illumina HiSeq 2500 system. RNA-seq reads were aligned with default settings. Count data were normalized. Genes were defined as differentially expressed if the fold change was less than 1.5 or less than 0.67, with an FDR less than 0.05, and at least one sample showing reads per kilobase per million mapped reads greater than 1. We performed hierarchical clustering of DEGs using Cluster v.3.0 with Pearson correlation distance and average linkage, and visualized them with Java TreeView. Enrichment analysis on the pathways of Hallmark, Kyoto Encyclopedia of Genes and Genomes and chemical genetic perturbation was performed using GSEA.

cGAMP levels were detected as reported elsewhere72,73. THP1 cells were DOX-treated to induce PRMT9 KD for 2 days; serum-free Phenol Red RPMI (Thermo Fisher Scientific) medium was replaced for another 24h. Conditioned medium was collected and cGAMP levels were detected using the Enzyme Immunoassay Kit (Arbo Assays). To determine cGAMP levels in the bone marrow microenvironment of control and Prmt9 KD mice, bone marrow fluid was collected by centrifuging tibias and femurs at 8,000rpm for 15s; then, cGAMP levels were assessed.

WT (catalog no. thpd-nfis, InvivoGen), cGAS KO (catalog no. thpd-kocgas, InvivoGen) and MAVS KO (catalog no. thpd-komavs, InvivoGen) THP1-Dual cells were used for the reporter assay. The purchased THP1-Dual cells (InvivoGen) were derived from the human THP1 monocyte line harboring the Lucia gene. Reporter cells were further engineered with inducible PRMT9 shRNA or control shRNA. After DOX treatment to PRMT9 KD or LD2 to inhibit PRMT9 in these cells, Lucia luciferase activity was determined as described by the manufacturer (InvivoGen) by adding QUANTI-Luc reagents and read with a FilterMax F5 microplate reader (Molecular Devices).

Cells were spun onto glass coverslips, fixed and incubated with primary anti-dsDNA (AE-2), H2AX or S9.6 antibodies, then with secondary antibody. Slides were then mounted in 90% glycerol solution containing DAPI (Thermo Fisher Scientific) and examined under a ZEISS LSM 880 confocal microscope.

We used the OxiSelect Comet Assay Kit (Cell Biolabs). Briefly, after PRMT9 KD, THP1 cells were mixed with prewarmed (37C) Comet agarose at a 1:10 ratio (v/v), then loaded onto the top of the Comet agarose base layer. Slides were immersed for 60min in lysis buffer at 4C, which was washed with prechilled alkaline solution. After three washes with prechilled Tris/Borate/EDTA buffer, slides were subjected to electrophoresis at 1Vcm1 for 15min, and then rinsed twice with deionized water. Comets were examined under a widefield ZEISS Axio Observer 7 fluorescence microscope. Approximately 50 cells were determined using the OpenComet software in Image J and shown as olive tail moments74,75.

THP1 reporter cells were electroporated with ribonucleoprotein complexes consisting of Cas9 protein and sgRNAs in the Neon Transfection System; 20moll1 guide RNA (gRNA) (as listed in Supplementary Table 11) were mixed at a 1:1 ratio. KO efficiency was assessed using immunoblot analysis.

As described previously57, bone marrow cells were cultured with complete RPMI medium containing 20ngml1 granulocyte-macrophage colony-stimulating factor (PeproTech). Fresh medium was added on days 3 and 6. CD8+ T cells were isolated from the spleens of OT-1 transgenic mice. MA9-OVA cells were pretreated for 2 days with LD2 and then cocultured overnight with collected bone marrow-derived DCs. Supernatants were collected for IFN- assessment. Bone marrow-derived DCs were selected using a CD11c+ selection kit (STEMCELL Technologies) and cocultured for 48h with OT-1 CD8+ T cells. IFN- supernatants were assayed using a mouse IFN- Flex Set Cytometric Bead Array.

Once leukemia cells were engrafted, MA9 syngeneic transplant mice were treated for 3 weeks with vehicle control, LD2, single anti-PD-1 mAb (catalog no. BE0146, Bio X Cell, 10mgkg1 intraperitoneally every other day) or LD2 plus anti-PD-1 antibody. LD2 was administered at 10mgkg1 intravenously twice a day, based on the preliminary pharmacokinetic and pharmacodynamic results. Mice were assessed for overall survival or killed directly to assess MA9 cell engraftment in bone marrow and perform staining with survivin-specific pentamers to assess MA9-specific immunity as described elsewhere44. Briefly, the bone marrow of MA9 mice was stained with anti-CD8 together with survivin-specific pentamers. CMV-specific pentamers were the negative controls. The percentage of survivin or CMV pentamer-positive CD8 T cells was assessed using flow cytometry. Secondary transplantations were performed to evaluate LSC activity in each group by assessing MA9 cell engraftment in the bone marrow.

The model was established using MHC class I and II DKO NSG mice49. To do so, we implanted 2 million MNCs from AML specimens intrafemorally into an irradiated DKO NSG mouse. After transplantation, MHC-deficient mice showed long-term (approximately 12 weeks in peripheral blood) engraftment of T and CD33+ cells without developing acute graft-versus-host disease. A panel of human lineage and progenitor cell markers (CD45, CD33, CD34, CD14, CD19, CD20, CD3, CD56, HLA-DR) was used to define T cells, B cells, monocytes, DCs and immature CD33+CD34+CD45dim cells. Mice were divided into two groups and treated with vehicle or LD2. Three weeks later, the number and frequency of leukemic CD34+ cells and the number of CD8+ T cells expressing CD69 and IFN- were assessed.

A20 cells (3106) were subcutaneously implanted into syngeneic BALB/c mice. When tumor volume reached 100mm3, mice were randomized into treatment groups. Tumor-bearing mice were treated with isotype control (vehicle), anti-PD-1 mAb (10mgkg1 intraperitoneally every other day for 2 weeks), LD2 (100mgkg1 intratissue injection daily for 2 weeks) or a combination of LD2 with anti-PD-1. Tumor volume was monitored through the end of the study when a humane endpoint was reached. The maximum tumor size (humane endpoint) permitted by the IACUC is 15mm (diameter). All animals were euthanized before tumor size reached 15mm in diameter. The microenvironmental components of tumors were analyzed using immunohistochemistry (IHC) and intracellular staining followed by flow cytometry.

Fixed A20 tumors were embedded in paraffin. Four-micrometer-thick sections on slides were incubated for 1h at 60C, deparaffinized and then rehydrated before IHC staining. Slides were blocked with 3% H2O2. Slides were subjected to antigen retrieval for 15min at 120C in citrate buffer, treated with Tris-buffered saline and incubated for 1h with anti-mouse CD3 or anti-mouse CD8 antibody. After washing, slides were incubated with secondary antibody. Slides were developed and counterstained with Mayers hematoxylin solution. Slides were scanned using whole slide imaging and analyzed using the NDP.view2 software (Hamamatsu).

Portions of fresh A20 tumors were cut into small pieces, then dissociated with type IV collagenase, type IV DNase and type V hyaluronidase at 37C for 30min. Cell suspensions were passed through a 70-m strainer and centrifuged at 300g for 5min. Cells were stained for 30min using a Live-or-Dye Fixable Viability Stain Kit (catalog no. 32018, Biotium). Next, cells were stained with immune cell surface markers (mouse CD45-allophycocyanin, mouse CD3-allophycocyanin/cyanine 7, mouse CD4-Alexa Fluor 700 and mouse CD8-Brilliant Violet 605). After two washes, cells were fixed and permeabilized, then intracellularly stained with mouse IFN--phycoerythrin and granzyme B-fluorescein isothiocyanate antibodies in the permeabilization for the flow analysis. Results were analyzed with FlowJo v.10 (FlowJo LLC).

Studies involving independent cohorts of mice were typically performed once, with several exceptions stated in the figure legends. No specific statistical tests were applied to determine sample size; size was established according to our previous experience with the models used. Accordingly, we typically used experimental cohorts of 57 mice. The experiments were not randomized. Investigators were not blinded to allocation during the experiments and outcome assessments. Data collection and analysis by all investigators were not performed blinded to the conditions of the experiments. No data were excluded from the analyses.

In general, data from independent experiments are shown as the means.d. or s.e.m. Statistics were determined using an unpaired, two-tailed Students t-test, a two-way ANOVA, a one-way ANOVA and a two-sided Fishers exact test. Survival results were analyzed with a log-rank (MantelCox) test and expressed as KaplanMeier survival curves. Prism (GraphPad Software) was used for the statistics; the detailed methods are described in each individual figure legend.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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Targeting PRMT9-mediated arginine methylation suppresses cancer stem cell maintenance and elicits cGAS-mediated ... - Nature.com

MD Anderson acquires inducible switch technologies for cell therapy – MD Anderson Cancer Center

The University of TexasMD AndersonCancer Center today announced it has acquired certain assets from Bellicum Pharmaceuticals, Inc. related to the CaspaCIDeswitch platform and the GoCAR platform. The transaction also includes clinical-grade stocks of rimiducid, an agent used to trigger the switches.

As a result of this acquisition, MDAndersonmay incorporate these platforms into its own cell therapy programs. The institution also intends to make the technology widely available via non-exclusive licenses to other academic institutions and to biopharmaceutical companies.

MD Anderson plans to focus on using the CaspaCIDe technology as a safety feature of cell therapies in development. The safety switch incorporates an inducible enzyme known as caspase-9, which initiates the first step of the apoptosis programmed cell death pathway. The switch can be triggered by rimiducid, leading to rapid elimination of cells containing the CaspaCIDe switch. MDAndersons Therapeutics Discovery division also plans to continue the clinical development of rimiducid in order to seek future approval from the Food and Drug Administration.

We strive each day to advance new, innovative treatment options to improve the lives of our patients, and cell therapies hold tremendous promise as effective immunotherapies, said Philip Jones, Ph.D., vice president of Research Strategy, Transformation and Operations atMDAnderson. CaspaCIDe provides a critical safety mechanism which could be triggered as required to reduce side effects, and we look forward to its continued development at MDAnderson.

Including this safety switch in cell therapies may offer clinicians the ability to quickly limit potential treatment-related toxicities that may occur. Potential applications include cell therapies where cytokine release syndrome and neurotoxicities have been observed, cell therapies targeting novel antigens with on-target/off-tumor safety concerns, and next-generation cell therapy constructs with higher potency.

When designing novel cell therapies, we must always ensure patient safety remains a top priority. We have explored a variety of associated technologies, and case studies demonstrate that the CaspaCIDe technology is effective in rapidly eliminating the transduced cells, saidKaty Rezvani, M.D., Ph.D., professor ofStem Cell Transplantation and Cellular Therapy. We have incorporated CaspaCIDe into many of our own cell therapies under investigation, and we are excited about the prospect of broadening the potential applications of this technology in the future.

Under a previous licensing agreement, MD Anderson has incorporated CaspaCIDe into multiple cellular therapy programs, including certain chimeric antigen receptor (CAR) natural killer (NK) cell therapies and plans for certain CAR T cell therapies. The current acquisition includes the previous licenses by MD Anderson and eliminates certain downstream financial obligations required under those licenses.

The asset acquisition also includes the transfer of certain intellectual property related to Bellicums GoCAR-T and GoCAR-NK technologies. Using the rimiducid-based switch system, GoCAR cell therapies feature an inducible MyD88/CD40 (iMC) activation switch designed to enhance proliferation and functional persistence of adoptive cell therapies by resisting exhaustion and by driving production of immunomodulatory cytokines to overcome inhibitory signals from the tumor microenvironment. GoCAR cell therapies may be particularly well-suited for use in solid tumors given their immune suppressive tumor microenvironment.

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MD Anderson acquires inducible switch technologies for cell therapy - MD Anderson Cancer Center