Archive for the ‘Induced Pluripotent Stem Cells’ Category

Induced pluripotent stem cells don’t increase genetic …

Induced Pluripotent Stem Cells | Posted by admin
Apr 30 2019

Called induced pluripotent stem cells (iPSCs), this technique opens the doors to medical advances, including generating cartilage cell tissue to repair knees, retinal cells to improve the vision of those with age-related macular degeneration and other eye diseases, and cardiac cells to restore damaged heart tissues.

Despite its immense promise, adoption of iPSCs in biomedical research and medicine has been slowed by concerns that these cells are prone to increased numbers of genetic mutations.

A new study by scientists at the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health, suggests that iPSCs do not develop more mutations than cells that are duplicated by subcloning. Subcloning is a technique where single cells are cultured individually and then grown into a cell line. The technique is similar to the iPSC except the subcloned cells are not treated with the reprogramming factors which were thought to cause mutations. The researchers published their findings on February 6, 2017, in the Proceedings of the National Academy of Sciences.

"This technology will eventually change how doctors treat diseases. These findings suggest that the question of safety shouldn't impede research using iPSC," said Pu Paul Liu, M.D., Ph.D., co-author, senior investigator in NHGRI's Translational and Functional Genomics Branch and deputy scientific director for the Division of Intramural Research.

Dr. Liu and his collaborators examined two sets of donated cells: one set from a healthy person and the second set from a person with a blood disease called familial platelet disorder. Using skin cells from the same donor, they created genetically identical copies of the cells using both the iPSC and the subcloning techniques. They then sequenced the DNA of the skin cells as well as the iPSCs and the subcloned cells and determined that mutations occurred at the same rate in cells that were reprogrammed and in cells that were subcloned.

Most genetic variants detected in the iPSCs and subclones were rare genetic variants inherited from the parent skin cells. This finding suggests that most mutations in iPSCs are not generated during the reprogramming or iPSC production phase and provides evidence that iPSCs are stable and safe to use for both basic and clinical research, Dr. Liu said.

"Based on this data, we plan to start using iPSCs to gain a deeper understanding of how diseases start and progress," said Erika Mijin Kwon, Ph.D., co-author and NHGRI post-doctoral research fellow. "We eventually hope to develop new therapies to treat patients with leukemia using their own iPSCs. We encourage other researchers to embrace the use of iPSCs."

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Induced Pluripotent Stem Cells – Embryo Project

Induced Pluripotent Stem Cells | Posted by admin
Apr 25 2019

Induced Pluripotent Stem Cells (iPSCs) are cells derived from non-pluripotent cells, such as adult somatic cells, that have been genetically manipulated so as to return to an undifferentiated, pluripotent state. Research on iPSCs, initiated by Shinya Yamanaka in 2006 and extended by James Thompson in 2007, has so far revealed the same properties as embryonic stem cells (ESCs), making their discovery potentially very beneficial for scientists and ethicists alike. By avoiding the destruction of embryos and the complicated technique and resource requirements of ESCs, iPSCs may prove more practical and attractive than ESC research in the study of pluripotent stem cells.

Yamanaka and his team were able to revert the differentiated cells to a pluripotent stage by using a retrovirus to insert specific genes known to be active in ESCs into the cells genome. Originally performed with mouse cells by Yamanaka and his team at Japans Kyoto University, both Yamanakas group and James Thompsons research team at University of Wisconsin, Madison, extended the technique to human somatic cells in November of 2007. A variety of genes and gene families have been identified as key components of a successful induction to the pluripotent state: Oct-3/4, the Sox family, the Klf family, the Myc family, Nanog, and LIN28. Additional genes that are expressed in ESCs include GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.

Unlike ESCs, iPSCs do not require embryos or even eggs from female donors a feature that has made them very appealing to scientists wishing to do work on pluripotent stem cells, which has heretofore been restricted in the United States and elsewhere due to ethical concerns and legal limitations. Though early work with iPSCs failed to produce living mice from embryos containing iPSCs, several research teams in the US and Japan achieved success after injecting iPSCs into developing embryos. The insertion of iPSCs into mice also originally caused high rates of cancerous tumors, but removal of the c-Myc genes from the retrovirus apparently eliminates the unusually high risk of cancer, according to further 2008 research by Yamanaka and his team.

Despite the genetic alteration involved in producing iPSCs, in most other aspects they are as yet indistinguishable from ESCs. In fact, the skills and resources required to produce iPSCs are significantly less labor-intensive and costly than those required for ESCs, in that most scientists with experience in genetic reprogramming can produce iPSCs. Neither iPSCs nor ESCs have yet been used in clinical treatments, though many researchers believe that undifferentiated cells hold even more potential for therapeutic applications than do adult stem cells, which are already used in a variety of therapies.

Immediately hailed by the media as the next step toward personalized medicine and the answer to the ESC research controversy, many researchers, ethicists, writers, and anti-ESC research groups have called for an end to or reduction in ESC research and funding. Scientists in the field, including some of the teams working with iPSCs, caution that it is still too soon to assume that iPSCs can replace ESCs in clinical potential and that ESC research will continue to be important in increasing sciences understanding of developmental biology. In addition, some scholars caution that iPSCs may eventually be altered to reach the totipotent state, which could nullify their ethical simplicity and place them on equal footing with embryos.

Though iPSCs show a great deal of potential for stem cell therapies and clinical applications, scientists are still in the fledgling research stages for this technology. If they surpass ESCs in practicality and success rates without totipotent capabilities, however, iPSCs may lay much of the ethical controversy surrounding ESC research to rest.

Brind'Amour, Katherine, "Induced Pluripotent Stem Cells".

(2010-05-06). ISSN: 1940-5030 http://embryo.asu.edu/handle/10776/1974.

Arizona State University. School of Life Sciences. Center for Biology and Society. Embryo Project Encyclopedia.

Arizona Board of Regents Licensed as Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported (CC BY-NC-SA 3.0) http://creativecommons.org/licenses/by-nc-sa/3.0/

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Induced Pluripotent Stem Cells - Embryo Project

What Are Induced Pluripotent Stem Cells? – Stem Cell: The …

Induced Pluripotent Stem Cells | Posted by admin
Apr 13 2019

Today, induced pluripotent stem cells are mostly used to understand how certain diseases occur and how they work. By using IPS cells, one can actually study the cells and tissues affected by the disease without causing unnecessary harm to the patient. For example, its extremely difficult to obtain actual brain cells from a living patient with Parkinsons Disease. This process is even more complicated if you want to study the disease in its early stages before symptoms begin presenting themselves.

Fortunately, with genetic reprogramming, researchers can now achieve this. Scientists can do a skin biopsy of a patient with Parkinsons disease and create IPS cells. These IPS cells can then be converted into neurons, which will have the same genetic make-up as the patients own cells.

Because of IPS cells, researchers can now study conditions like Parkinsons disease to determine what went wrong and why. They can also test out new treatment methods in hopes of protecting the patient against the disease or curing it after diagnosis.

In addition, IPS cells have also been looked to as a way to replace cells that are often destroyed by certain diseases. However, there is still research to be done here.

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Gene therapy-corrected autologous hepatocyte-like cells …

Induced Pluripotent Stem Cells | Posted by admin
Apr 03 2019

Our research group has been able to successfully develop induced pluripotent stem cells from patients with arginase deficiency, a poorly treated metabolic disorder of the liver that results in intellectual disabilities. In this disorder, the enzyme argianse is mutated and does not function in a normal process called the urea cycle which handles nitrogen metabolism. An excess of nitrogen in the body (through netabolism, diet, injury, or illness), typically as ammonia, can cause brain injury. We have been able to successfully introduce a normal copy of the arginase gene into induced pluripotent stem cells  from the patient-derived cells and then developed them into hepatocyte-like cells. At the same time, we have been utilizing a mouse model of arginase deficiency that was further modified to carry genes that suppress their immune system. We have been able to repopulate the liver of these mice with normal human hepatocytes that has led to correction of the defect related to arginase deficiency. These animals demonstrate almost normal circualting blood levels of arginine and ammonia and have improved handling of nitrogen when it is delivered to the mice as an ammonia injection. At present, the induced pluripotent-derived hepatocytes have not engrafted in these mice,and  we are continuing to work on strategies to lead to engraftmentment and thus treatment of this disorder. The ultiamte goal of treatment is to be able to take skin cells from a patient with this disorder, develop them into stem cells, add a corrected copy of the arginase gene, develop these cells into hepatocytes and deliver them to the same patient's liver to correct the disorder. WIth the data to date we are on our way to achieving this goal.

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Regenerative Potential Of Induced Pluripotent Stem Cells …

Induced Pluripotent Stem Cells | Posted by admin
Mar 28 2019

1020 0 Posted on Mar 26, 2019, 9 p.m.

After the discovery of induced pluripotent stem cells in the Nobel Prize winning lab of researcher Shinya Yamanaka at Kyoto University in 2006, the thick morality lined clouds hovering over embryonic stem cell research began to fade away making room for happier times.

Stem cells harvested from adults cells rather than embryos are less controversial and hold some promise for practical benefits as well such as lower chance of rejection by the immune system as they can be created from the patients very own cells. iPSCs are a versatile tool which is proving to be useful in modeling disease, screening drugs, and holds massive promise in the field of regenerative medicine.

Osaka and Cardiff Universities explored how iPSCs may be able to restore vision in humans. Using stem cells they were able to generate multiple cells lineages that resulted in tissues being implanted into the eyes of rabbits with induced corneal blindness which repaired the organ and restored their vision. This demonstrates various types of human stem cells are able to take on characteristics of the cornea, lens, and retinas which paves the way for trials to explore the technology in humans. according to Professor Andrew Quantock.

Kyoto University researchers explored the idea of using iPSCs to arrest the slide in decline of dopamine which hampers motor skills in those with Parkinsons disease, using diseased monkey brains with cells converted into dopaminergic progenitors responsible for generating dopamine neurotransmitters. The research has gone so well they are now conducting human trials using the same technology; 7 Parkinsons disease patients were given 5 million iPSC derived dopaminergic progenitors which were transplanted directly into their brains with a special device in hopes that it will curtail effects of the disease. The patients are being closely monitored and observed over the next two years.

University of Minnesota researchers have created a cutting edge technology wherein cells can be converted into neural stem cells, which can be mixed and matched with alternating layers of silicon scaffold to be used to grow new connections in the spine between the nerves that remain in spinal cord injury. In lab testing this technology was found to grow nerves and connect undamaged separated cells. Testing showed new neurons could be grown in an injury site, however the work is still a way off in doing so in numbers that would allow a paralyzed human to walk again. Even with that being said partial repairing of the spinal cord could still improve functions such as bladder control, avoid involuntary movement of limbs, and improve quality of life which is still plenty reason to be excited.

Washington University research has made a breakthrough with a method of converting iPSCs into beta cells which were tested by implanting into diabetic mice incapable of producing insulin on their own. The animals began secreting insulin on their own within days in quantities sufficient to control their own blood sugar levels which functionally cured their condition.

Following the successful trials on pigs in 2017 the Japanese government has recently approved the first ever trial on humans wherein iPSCs are being used to create sheets bearing millions of heart muscle cells which are then grafted onto the heart of human patients with heart disease. It is hoped with the help of growth factors these sheets will promote regeneration of damaged muscles and improve the function of the heart. This first of its kind trial involves 3 patients, if all goes well the team hopes to have a larger trial with 10 patients, followed by commercial availability of the technique if all goes according to plan.

There is no shortage of research into male pattern baldness, recently we are being shown how stem cells may play a role in hair revival, such as wherein scientists had converted iPSCs into epithelial stem cells which gave rise to hair follicle on the skin of immunodeficient mice. Another technique coaxed iPSCs into the form of dermal papilla cells which were transplanted into mice which triggered new hair growth. Research from the University of Southern California harvested skin cells from adults, molecular events behind their growth was examined and then replicated in iPSCs to grow new hair follicles in mice.

iPSCs are opening up a diverse array of exciting new medical possibilities. At only 13 years of discovery this very well is likely just the tip of the iceberg of many wonderful things to come. What a great time this could be for the future of the entire medical field and the possibility of regenerative medicine.

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Induced Pluripotent Stem Cell (iPSC) Media and Reagents for …

Induced Pluripotent Stem Cells | Posted by admin
Mar 21 2019

Advancing your induced pluripotent stem cells or human embryonic stem cell therapy research to clinical applications requires careful material selection because the quality of starting materials significantly impact the properties of your final stem cell therapy product. Gibco CTS products have been developed to ease the transition from stem cell therapy research to clinical applications by providing high quality GMP manufactured, commercial scale ancillary materials with a high degree of qualification, traceability and regulatory documentation. In an effort to help you maximize the potential of your stem cell research and therapy, and simplify the transition to clinic-ready processes, we offer an extensive selection of research use stem cell research products with complementary CTS formulations. Our CTS products are used in commercially approved cell therapies as well as over 100 clinical trials and are backed by our professional regulatory support and over 30 years of GMP manufacturing experience.

Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) , sometimes collectively referred to as pluripotent stem cells (PSCs), are cells that have the ability to renew themselves indefinitely and differentiate into almost any cell type when exposed to the right microenvironment. These unique properties enable the application of induced pluripotent stem cells and embryonic stem cells in disease modeling, drug discovery, drug toxicity testing, and cell therapy. Strikingly, most embryonic stem cell and induced pluripotent stem cell applications have the potential to improve human health, none more directly so than ESC or iPSC therapy. The most intuitive approach for ES or iPS cell therapy is to transplant PSC-derived cells for the direct replacement of damaged or degenerated cells or tissue. However, there are many other approaches to ES or iPS stem cell therapy such as transplanting PSC-derived cells that then release signals triggering endogenous repair mechanisms.

At Thermo Fisher Scientific, we support the development of your human embryonic stem cell therapy or induced pluripotent stem cell therapy from the earliest stages of research and all the way to the clinic. We offer high-quality products across the iPS cell therapy workflow from reprogramming to differentiation. Most Gibco media and supplements for culture and differentiation are manufactured under GMP conditions at sites that use methods and controls that conform to current Good Manufacturing Practices (cGMP) for medical devices. These FDA-registered manufacturing sites are ISO 13485 and ISO 9001certified, and the rigorous practices we adhere to at these sites help ensure the consistency, reliability, and high quality of a wide variety of iPSC therapy workflow reagents.

To further help you maximize the potential of your research and streamline your transition to the clinic, we offer Gibco Cell Therapy Systems (CTS) equivalents for many of our research-use products. In addition to GMP manufacturing, Gibco CTS products undergo extensive safety testing and are accompanied by appropriate documentation so you can transition your cell therapy to the clinic with confidence.

*Adherence to supplier related responsibilities of USP

First off-the-shelf reprogramming system manufactured in accordance with GMP requirements. CTS CytoTune 2.1 kit offers high-efficiency Sendai delivery of reprogramming factors.

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Efficient reprogramming from adult human dermal fibroblasts, T cells, and CD34+ cells. These data demonstrate that the CytoTune-iPS 2.1 kit can be used to successfully reprogram human dermal fibroblasts (HDFa), T cells, and CD34+ cells.

Based on the widely cited Gibco Essential 8 Medium, Gibco CTS Essential 8 Medium is the first globally available human- and animal originfree culture medium for human pluripotent stem cells (hPSCs) and is designed to meet international regulatory requirements for cell therapy.

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Using Applied Biosystems TaqMan hPSC Scorecard Panel analysis, Gibco CTS Essential 8 Medium and research-use-only Essential 8 Medium were shown to support comparable expression of PSC markers and lineage markers in undifferentiated PSCs and PSC-derived embryoid bodies.

CTS Vitronectin (VTN-N) Recombinant Human Protein is a defined matrix for feeder-free culture of iPSCs. Designed in the laboratory of James Thomson, this recombinant protein is intended for use with the CTS Essential 8 culture system.

CTS RevitaCell Supplement (100X) is an animal-origin-free, chemically defined supplement used with PSCs for post-thaw recovery or in combination with CTS Essential 8 Medium for single cell passaging. To minimize both the loss of cell viability and differentiation of PSCs, use the CTS PSC Cryopreservation Kit.

CTS Versene is a gentle non-enzymatic cell dissociation reagent for use in routine clump passaging of PSCs while maintaining viability over multiple passages.

For the cryopreservation and recovery of PSCs, the CTS PSC Cryopreservation Medium and CTS RevitaCell Supplement minimize the loss of cell viability and maximize post-thaw recovery when used in combination. Both reagents are included in the CTS PSC Cryopreservation Kit.

The CTS PSC Cryopreservation Medium is a xeno-free solution for the cryopreservation of pluripotent stem cells (PSCs). Both CTS PSC Cryopreservation Medium and CTS RevitaCell supplement are included in the CTS PSC Cryopreservation Kit that helps minimize loss of cell viability and maximize post-thaw recovery.

CTS KnockOut SR XenoFree Medium is a defined, xeno-free serum replacement based on the traditional Gibco KnockOut Serum Replacement, which has been cited in more than 2,000 publications and trusted for over 20 years.

Maintenance of pluripotency using CTS KNOCKOUT SR XenoFree Medium. Following 10 passages in either KSR (left lane) or KSR XenoFree CTS (right lane) on HFF attached with CELLstart substrate, BG01v gene expression was examined (top). Gene expression of embryoid bodies generated from the same P10 BG01v/HFF cultures (bottom).

Your choice of chemically defined human- and animal origin-free basal media for pluripotent stem cell culture. Based on traditional DMEM and DMEM/F12 formulations, these basal media are:

Gibco CTS growth factors help enable you to easily qualify reagents during your transition from research applications to clinical applications. CTS products are supplied with harmonized documentation such as Certificates of Analysis and Certificates of Origin.

CTS offers high-quality growth factors and cytokines for T cell, stem cell and dendritic cell applications.

CTP0261,CTP0263,CTP9211,CTP9213,CTP2111,CTP2113,CTP9411,CTP9413

We offer full customization options to help meet your unique specifications for any project. Flexibility is yours in creating your own Gibco custom cell culture medium

Intended use of the products mentioned on this page vary. For specific intended use statements please refer to the product label.

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Embryonic or Induced Pluripotent Stem Cell Markers: R&D Systems

Induced Pluripotent Stem Cells | Posted by admin
Mar 11 2019

Overview

Embryonic stem cells (ESCs) have the exceptional ability to both self-renew and differentiate into nearly every cell of the human body. Induced pluripotent stem cells (iPSCs) are somatic cells that have been reprogrammed back into an ESC-like phenotype. Both ESCs and iPSCs have numerous roles to play in drug discovery studies, understanding mechanisms of disease, cell therapies, and developmental biology.

Expression of Pluripotency Markers in Human Embryonic Stem Cells. Pluripotency marker expression was detected in immersion-fixed BG01V human embryonic stem cells using antibodies supplied in the Human Pluripotent Stem Cell Markers Antibody Panel (R&D Systems, Catalog # SC008). Pluripotency marker expression was analyzed by dual immunofluorescence with the indicated primary antibodies supplied in the panel. The cells were stained using NorthernLights (NL) 493- and NL557-conjugated Secondary Antibodies (green and red, respectively). Where indicated, the nuclei were counterstained with DAPI (blue).

Verification of Pluripotency in Human Induced Pluripotent Stem Cells. iPS2 human induced pluripotent stem cells were grown on irradiated mouse embryonic fibroblasts (R&D Systems, Catalog # PSC001) and stained using antibodies included in the GloLIVE Human Pluripotent Stem Cell Live Cell Imaging Kit (R&D Systems, Catalog # SC023B). A. iPS2 cells stained with the NL493-conjugated SSEA-4 (green) and the NL557-conjugated SSEA-1 (red) antibodies. B. iPS2 cells stained with the NL493-conjugated SSEA-4 (green) and the N557-conjugated TRA-1-60(R) (red) antibodies. The cells were counterstained with Hoechst 33342 (blue). The colonies are positive for the stem cell markers SSEA-4 and TRA-1-60(R) and are negative for SSEA-1, suggesting that these colonies primarily contain undifferentiated human stem cells.

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Embryonic or Induced Pluripotent Stem Cell Markers: R&D Systems

Differentiation of human induced pluripotent stem cells into …

Induced Pluripotent Stem Cells | Posted by admin
Mar 08 2019

Culture of human iPSCs

The commercial human iPSCs used in this study were purchased from Saibei Biotechnology (Beijing, China). iPSCs (HiPSC-U1) were reprogrammed from human urine-derived cells of a 37-year-old male by the integration-free CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, MA, USA). iPSCs were cultured in 1% Matrigel-coated (BD Biosciences Co., Ltd., NM, USA) Petri dishes with E8 medium (Saibei Biotechnology) at 37C and 5% CO2. The medium was refreshed daily. iPSCs were passaged once every 6 days with 0.25% ethylenediaminetetraacetic acid (EDTA, Saibei Biotechnology). 1mL of 0.25% EDTA was added and the cells were placed at 37C and 5% CO2 for 5min. When iPSC colonies appeared white, the solution was gently removed, and the iPSCs were washed with Mg2+ and Ca2+-free Dulbeccos PBS (Sigma, MO, USA). iPSCs were then harvested by gently pipetting 710 times with 1mL E8 medium and seeded onto fresh six-well culture plates that were coated with 1% Matrigel at a ratio of 1:6. 10m Y-27632 (Sigma) was supplemented in the medium on the first day of passage.

Human LCs were obtained from nine male donors with a mean age of 45 years old through testes excision within 20h. Informed consent was obtained from each donor, and this study was approved by Human Research and Ethical Committee of Wenzhou Medical University. The testes were used to isolate ILCs. ILCs express all androgen biosynthetic enzymes56, and are capable of proliferation and differentiation57. The isolation of ILCs was performed as previously described56. In brief, the testes were perfused with collagenase (Sigma) via the testicular artery, and digested with M-199 buffer (Gibco, NY, USA) containing collagenase (0.25mg/ml) and DNase (0.25mg/mL, Sigma) for 15min. Then, the cell suspension was filtered through 100m nylon mesh and the cells were separated by a Percoll gradient (Sigma). The cells with the density of 1.071.088g/ml were collected. The purity of ILCs was evaluated by immunohistochemical staining HSD3B1, the biomarker of ILCs, as previously described58. The HSD3B1 staining solution contained with 0.4mm etiocholanolone (Sigma) as the steroid substrate and NAD+ as a cofactor58. The purity of ILCs was >95%.

The isolated ILCs were directly seeded into wells in the 24-well culture plates with the density of 2104 cells/well and incubated at a 37C, 5% CO2 incubator. The culture medium (LC-Medium) contains DMEM/F12 (Gibco), 5% fatal bovine serum (FBS, Gibco), 2.5% horse serum (HS, Gibco), and 1% penicillin/streptomycin (P/S, Gibco). In order to get ALCs, the culture medium were changed into differentiation-induced medium (DIM) contains DMEM/F12, 5mm ITS (insulin, transferrin, and selenium, Sigma), 5ng/ml luteinizing hormone (LH, PeproTech, NJ, USA), and 5mm lithium chloride (Li, Sigma) as our team previous report35.

The Sprague-Dawley rats (at 5 weeks of age) and immune deficiency (SCID) mice (at 5 weeks of age) were obtained from the laboratory animal center of Wenzhou Medical University, Wenzhou, China. All animals were kept under conditions with controlled temperature (232C), a 12h dark/light cycle, and relative humidity of 4555%. The standard drinking water and rodent diet were accessed ad libitum. All surgical procedures and postoperative care were approved by the Wenzhou Medical Universitys Animal Care and Use Committee, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

The point at which iPSCs were expanded to ~70% confluency in the E8 medium was defined as day 2, and at this point when iPSCs were changed into E7 medium (no FGF2) for 2 days to prepare differentiation. From day 5 to 0, the medium was refreshed daily. Prior to the beginning of differentiation, iPSCs were cultured in a differentiation-inducing medium composed of DMEM/F12, 1% bovine serum albumin (BSA) (Sigma), 5mm ITS, 5ng/mL LH. From 07 days, 0.2m SAG (DHH agonist, Sigma), 5m 22R-OHC (Steraloids, RI, USA), and 5mm Li were added into iPSC-DIM. From 710 days, 5ng/mL PDGF-AA (Sigma) and 5ng/mL FGF2 (Sigma) were added into iPSC-DIM. From 1017 days, 5ng/mL PDGF-AA, 5nM IGF1 (Sigma), and 10m Androgen (Sigma) were added into iPSC-DIM. From 1720 days, 10ng/mL PDGF-AA and 10ng/mL FGF2 were added into iPSC-DIM. From 2025 days, 5ng/mL LH, 0.5mm retinoic acid (RA, Sigma) and 1mm 8-Br-cAMP (Sigma) were added into iPSC-DIM. From day 0 to 25, the medium was changed every 2 days by fresh iPSC-DIM. From 2530 days, the cells were mechanically enriched by scraping away clonal iPSC-like cells. The remaining Leydig-like cells were kept in Enrichment Medium contained DMEM/F12, 5% FBS, 2.5% HS, 1sodiumpyruvate (Invitrogen), 1GlutaMAX (Invitrogen), and 1% P/S for the subsequent assays. The medium was changed every 2 days by fresh Enrichment Medium.

For TEM, the cells in different groups were prefixed with 2.5% glutaraldehyde in 0.1m PBS for 24h at 4C. Then, they were washed with PBS, and post-fixed with 1% osmium tetroxide. After gradient dehydration of acetone, they were embedded in Araldite M (Sigma Aldrich). Ultrathin sections (1m) were subsequently cut with an ultramicrotome, mounted on nickel grids, and stained with uranyl acetate and lead citrate. At last, the samples were sent to the electron microscope room at Wenzhou Medical University for subsequent processing and testing using a transmission electron microscope (H-600A-2; Hitachi, Tokyo, Japan).

The cell culture supernatants and serum were collected at each experimental time point for the quantitative measurement of testosterone. For the cell culture supernatants, 10ng/mL LH was in advance at least 3h added into the medium (just having DMEM/F12) to stimulate the testosterone production of LCs or iPSC-LCs. Testosterone levels were measured with a tritium-based radioimmunoassay using anti-testosterone antibody as previously described59. Standards ranging between 10 and 2000pg/mL testosterone were prepared in triplicate. Standards and samples were incubated with tracer and antibody at 4C overnight and charcoal-dextran suspension was used to separate the bound and free steroids. The bound steroids were mixed with a scintillation buffer and counted in a scintillation counter (PE, CA, USA). The minimum detectable concentration for testosterone was 5pg/mL. Quality control samples contain 100pg/mL testosterone. The intra-assay and inter-assay coefficients of variation were within 10%.

Immunofluorescence was used to identify iPSC-LCs as a previous report60. In brief, after fixation with 4% paraformaldehyde (Sigma) for 15min, cells were washed three times with PBS. Then cells were permeabilized with 0.1% TritonX-100 in PBS for 15min at room temperature, and incubated with 3% (w/v) BSA in PBS for 1h at room temperature. The cells were then incubated with primary antibodies as TableS1 overnight at 4C, and then with fluorescein isothiocyanate (FITC)-conjugated anti-mouse, FITC-conjugated anti-rabbit, Cy3-conjugated anti-mouse, and Cy3-conjugated anti-rabbit IgG secondary antibodies (1:1000, Bioword, USA) for 60min at room temperature. Then the cells were rinsed three times with PBS thrice for 5min each and then incubated for 15min with DAPI (Sigma) for nuclear staining and washed three times with PBS before examination by an inverted fluorescence microscope (OLYMPUS, Japan).

Total RNA from the cells was extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturers instruction. The RNA was reversely transcribed into cDNA using the Superscript II kit (Invitrogen). The cDNAs templates were diluted 1:10, which were used to perform RT-PCR and qPCR to analyze the gene expressions. RT-PCR was performed using an authorized thermal cycler (Eppendorf, Hamburg, GER). After amplification, 1L of 6Loading buffer and 5L of each PCR product were mixed and electrophoresed on a 2% agarose containing 0.5g/mL ethidium bromide. Gels were scanned for further analysis. qPCR was performed using the Thunderbird SYBR qPCR Mix (Takara, Tokyo, Japan) according to the manufacturers instructions. Signals were detected using a Light Cycler 480 Detection System (Roche, Basel, Switzerland). The relative expression of genes was normalized to GAPDH. The melting curve was examined for the quality of PCR amplification for each sample, and quantification was performed using the comparative 2-Ct method. The primer sequences were shown in TableS2.

Total RNA from each sample was extracted using Trizol reagent (Invitrogen, CA, USA). 12g total RNA was used to prepare the sequencing library. To sequence the libraries, the barcoded libraries were mixed, denatured to single stranded DNA, captured on Illumina flow cell, amplified in situ, and subsequently sequenced for 150 cycles for both ends on Illumina HiSeq 4000 instrument. Sequence quality was examined using the FastQC software. The transcript abundances for each sample were estimated with StringTie, and the FPKM value for gene and transcript level were calculated with R package Ballgown. The differentially expressed genes and transcripts were filtered using R package Ballgown. The correlation analysis was based on gene expression levels. Hierarchical Clustering, Gene Ontology, Pathway analysis, scatter plots and volcano plots were performed with the differentially expressed genes in R, Python for statistical computing and graphics61.

Cells were washed with cold PBS and were lysed in 1radioimmunoprecipitation assay lysis buffer in the presence of a protease inhibitor mixture/1% phosphatase inhibitor mixture (Roche). 50g of protein samples were applied to a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred into the polyvinylidene difluoride membranes (Sigma) by an electroblot apparatus. After being blocked with a blocking solution (5% fat-free milk) for 2h at 4C, the membranes were incubated with primary antibodies as TableS1 in the blocking solution at 4C overnight. The membranes were washed with tris-buffered saline with Tween 20 (TBST) five times (10min each), and incubated with horseradish peroxidase-conjugated secondary antibody (1:3000, Bioword) at room temperature for 2h. The membranes were then washed five times (10min each) with TBST. Bands were visualized with enhanced chemiluminescence (ECL, Pierce, USA). The protein expression was normalized to -actin.

The cell samples were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% TritonX-100 (Sigma). The samples were then labeled with primary or isotype control antibodies for 30min at 4C. Primary and isotype control antibodies that were not conjugated to fluorophores were labeled with fluorophore-conjugated secondary antibody for 30min at 4C. The labeled samples were detected by flow cytometry analyzer (BD, USA). Data analysis was performed on FCS Express 4 Flow Research Edition software.

The standard protocol was conducted as PKH26 Product Information Sheet (Sigma, MINI2). In brief, the suspension containing 2107 cells were centrifuged (400g, 5min) and were washed once using fresh medium without serum. After centrifuging, the supernatant was removed and no more than 25L of 2Cell Suspension was prepared by adding 1mL of Diluent C, the cells were resuspended with gentle pipetting to ensure complete dispersion. 2Dye Solution (4106m) was prepared by adding 4L of the PKH26 ethanolic dye solution to 1mL of Diluent C and mixed well. Then 1mL of 2Dye Solution was rapidly added into the 1mL of 2Cell Suspension. Final concentration after mixing was 2106m PKH26 with 1107 cells/well. The mixing suspension was incubated at room temperature for 5min with periodic mixing. The action of the staining was stopped by adding an equal volume (2mL) of serum. Then the suspension was centrifuged at 400g for 10min and washed three times. Finally, the cells tagged with PKH26 were seeded on fresh wells and used for injection.

For evaluating whether iPSC-LCs could facilitate the recovery of LC dysfunction of rats, iPSC-LC transplantation was performed as previously described with some modifications62. Sixty 49-days-old male Sprague-Dawley rats (n=5 for each group at each time point) were used in this study. Before transplantation, male rats were administered a single intraperitoneal injection of EDS (75mg/kg, Pterosaur Biotech Co., Ltd., Hangzhou, China), which was dissolved in dimethyl sulfoxide (Sigma): H2O (1: 3, v/v). This treatment resulted in the elimination of LCs in the adult testes of rats63. Then iPSC-LCs labeled with PKH26 (red) were resuspended manually, and harvested in a 15mL Falcon tube. Cells were rinsed twice with PBS following centrifugation at 200g for 5min. Finally each pellet was resuspended in PBS for transplantation. Cells were loaded into a 1mL syringe for injection into the testis of adult Sprague-Dawley male rats that had been treated with EDS. Approximately 2106 PKH26-labeled iPSC-LCs in 40mL of PBS were injected into the parenchyma of recipient testes 7 days after the rats received EDS. The control animals for the experimental group were EDS-treated rats that had received a testicular injection of the PBS vehicle. Testes from all animals were examined at 0, 7, 14, and 21 days after EDS treatment.

One testis from each rat was used for immunohistochemistry (Vector Laboratories, Inc., Burlingame, CA, USA) according to the manufacturers instructions. The rats were killed with an overdose of sodium pentobarbital (Sigma). Testes were removed and fixed in 4% paraformaldehyde overnight at 4C. Then testes were dehydrated with a graded series of ethanol and xylene and subsequently embedded in paraffin. Five micrometer-thick transverse sections (5m) were cut, de-waxed in water, and were mounted on glass slides. Antigen retrieval was performed by microwave irradiation for 10min in 10mm (pH 6.0) of citrate buffer, after which endogenous peroxidase was blocked with 0.5% of H2O2 in methanol for 30min. Some sections were fixed with 4% paraformaldehyde for 15min and washed 3 times with PBS. Then they were permeabilized with 0.1% TritonX-100 in PBS for 15min at room temperature, and incubated with 3% (w/v) BSA (Sigma) in PBS for 1h at room temperature. Then these sections were then incubated with an CYP11A1 polyclonal antibody diluted 1:1000 for 2h at room temperature, and then with FITC-conjugated IgG secondary antibodies (1:1000, Bioword) for 1h at room temperature. These sections were rinsed with PBS three times for 5min each time. Then the sections were incubated for 15min with DAPI (10g/mL, Sigma) for nuclear staining and washed three times with PBS. The sections were cover-slipped with resin (Thermo Fisher Scientific, Waltham, UK). At last, they were examined by an inverted fluorescence microscope (OLYMPUS). The cells with CYP11A1 staining in the interstitial area represent the LC64.

Other sections were directly incubated with CYP11A1 polyclonal antibody diluted 1:1000, for 2h at room temperature. Diaminobenzidine was used for visualizing the antibodyantigen complexes, positive labeling LCs by brown cytoplasmic staining. Mayer hematoxylin was applied in counterstaining. The sections were then dehydrated in graded concentrations of alcohol and cover-slipped with resin (Thermo Fisher Scientific, Waltham, UK). Lastly, they were examined by a fluorescence microscope (LEICA). The cells with CYP11A1 staining in the interstitial area represent the LCs.

For teratoma formation, iPSCs (5106 cells) were dissociated with 0.5mm EDTA, centrifuged, resuspended in 100L E8 with 1% Matrigel, and injected into the hind limbs of 6-week-old male SCID mice. Teratomas were collected after 6 weeks, and fixed in 4% paraformaldehyde for paraffin embedding and hematoxylin and eosin staining. Slides were imaged and analyzed by a qualified clinical pathologist.

The division of iPSCs was blocked with 50g/mL of colcemid solution (Invitrogen, USA). Cells were washed with PBS and harvested with trypsin at room temperature for 2min. Then cells were fixed in methanol/glacial acetic acid (3:1) for three times and dropped onto slides for chromosome spreads. At last, the slides were baked at 55C for overnight. Standard G-banded karyotypes were obtained using Giemsa solution staining (Giemsa, Japan).

To enumerate CYP11A1-positive Leydig cell numbers, sampling of the testis was performed according to a fractionator method as our previous report65. Identification of all Leydig cell lineages was done by the staining of CYP11A1. About 10 testis sections per rat were sampled from each testis. The total number of LCs was calculated by multiplying the number of LCs counted in a known fraction of the testis by the inverse of the sampling probability.

All experiments were performed at least thrice, and the data are presented as the meanstandard error of the mean. Statistical analyses were evaluated using an unpaired Students t test or one-way analysis of variance for more than two groups. P

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Differentiation of human induced pluripotent stem cells into ...

Do You Know the 5 Types of Stem Cells? | BioInformant

Induced Pluripotent Stem Cells | Posted by admin
Mar 04 2019

*Post also available in: Espaol Romn

As you start to learn about stem cells, one of the most common questions tohave is, What types of stem cells exist?There is not an agreed-upon number of stem cell types, because one can classify stem cells either by differentiation potential(what they can turn into) or by origin (from where they are sourced).This post is dedicated to explaining the five types of stem cells, based on differentiation potential.

The five different types of stem cells discussed in this article are:

All stem cells that exist can be classified into one of five groups based on their differentiation potential. Each of these stem cell types is explored in greater detail below.

The Rise of Direct Cell Reprogramming | BioInformant https://t.co/q0vwT6CffR#allogeneic #totipotent #pluripotent #multipotent #autologous pic.twitter.com/ycoDP8mYa6

Todd C Bertsch (@todd_bertsch) February 19, 2018

These stem cells are the most powerful that exist.

They can differentiate into embryonic, as well as extra-embryonic tissues, such as chorion, yolk sac, amnion, and the allantois. In humans and other placental animals, these tissues form the placenta.

The most important characteristic of a totipotent cell is that it can generate a fully-functional, living organism.

The best-known example of a totipotent cell is a fertilized egg (formed when a sperm and egg unite to form a zygote).

It is at or around four days post-fertilization that these cells begin to specialize into pluripotent cells, which as described below are flexible cell types, but cannot produce an entire organism.

Theyre aliveeee!! Turned our human pluripotent stem cells into beating cardio!!! ::happy tears:: Next up crispR KO fun #stemcellscientist #WomenInScience #futureBIOhacker pic.twitter.com/GVg4pb9Xri

Kristin Pagel (@DeeDeeTroit84) March 31, 2018

The next most powerful type of stem cell is the pluripotent stem cell.

The importance of this cell type is that it can self-renew and differentiate into any of the three germ layers, which are: ectoderm, endoderm, and mesoderm. These three germ layers further differentiate to form all tissues and organs within a human being.

There are several known types of pluripotent stem cells.

Among the natural pluripotent stem cells, embryonic stem cells are the best example.However, a type of human-made pluripotent stem cell also exists, which is the induced pluripotent stem cell (iPS cell).

iPS cells were first produced from mouse cells in 2006 and human cells in 2007, and are tissue-specific cells that can be reprogrammed to become functionally similar to embryonic stem cells.

Because of their powerful ability to differentiate in a wide diversity of tissues and their non-controversial nature, induced pluripotent stem cells are well-suited for use in cellular therapy and regenerative medicine.

Did you know that bone marrow contains multipotent stem cells that give rise to all the cells of the blood? pic.twitter.com/NcYJsdPJXi

caremotto (@caremotto) January 17, 2018

Multipotent stem cells are a middle-range type of stem cell, in that they can self-renew and differentiate into a specific range of cell types.

An excellent example of this cell type is the mesenchymal stem cell (MSC).

Mesenchymal stem cells can differentiate into osteoblasts (a type of bone cell), myocytes (muscle cells), adipocytes (fat cells), and chondrocytes (cartilage cells).

These cells types are fairly diverse in their characteristics, which is why mesenchymal stem cells are classified as multipotent stem cells.

The next type of stem cells, oligopotent cells, are similar to the prior category (multipotent stem cells), but they become further restricted in their capacity to differentiate.

While these cells can self-renew and differentiate, they can only do so to a limited extent. They can only do so into closely related cell types.

An excellent example of this cell type is the hematopoietic stem cell (HSC).

HSCs are cells derived from mesoderm that can differentiate into other blood cells. Specifically, HSCs are oligopotent stem cells that can differentiate into both myeloid and lymphoid cells.

Myeloid cells includebasophils, dendritic cells, eosinophils, erythrocytes, macrophages, megakaryocytes, monocytes, neutrophils, and platelets, while lymphoid cells include B cells, T cells, and natural kills cells.

Finally, we have the unipotent stem cells, which are the least potent and most limited type of stem cell.

An example of this stem cell type would be muscle stem cells.

While muscle stem cells can self-renew and differentiate, they can only do so into a single cell type. They are unidirectional in their differentiation capacity.

The purpose of these stem cellcategories is to assess thefunctional capacity of stem cells based on their differentiation potential.

Importantly, each category has different stem cell research applications, medical applications, and drug development applications.

Watch this video and learn about the 5 types of stem cells:

If you found this article valuable, subscribe to BioInformants stem cell industry updates.

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In your opinion, which of the following types of stem cells have the best potential to form any tissue type? Mention them in the comments section below.

To learn more, view:Stem Cell Fact Sheet Types of Stem Cells and their Use in Medicine

Do You Know The 5 Types Of Stem Cells?

Cade Hildrethis the Founder ofBioInformant.com, the world's largest publisher of stem cell industry news.Cade is a media expert on stem cells, recently interviewed by theWall Street Journal,Los Angeles Business Journal, Xconomy,andVogue Magazine.

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Do You Know the 5 Types of Stem Cells? | BioInformant

Global transcriptome analysis of pig induced pluripotent …

Induced Pluripotent Stem Cells | Posted by admin
Mar 04 2019

The progress of next-generation sequencing technology has caused a technological breakthrough at the whole-genome level in a large number of species1. Especially, RNA-sequencing (RNA-Seq) has enabled us to take a snapshot of global gene expression in various organs and cells, regardless of any information of a reference genome. RNA-Seq outputs are digital data that can be uploaded to the public database, and sequence information can be shared worldwide.

RNA-Seq analysis also allows us to compare the biological similarity of embryonic stem cells (ES cell) with induced pluripotent stem (iPS) cells. In general, stem cells can be classified into two major subtypes: nave and primed states2,3. ES/iPS cells at nave state of pluripotency, reflect the characteristics of pre-implantation embryos and are applicable in rodents, which contribute to chimeras and germ line4,5. The growth of nave stem cell depends on the activation of LIF (Leukemia Inhibitory Factor) signaling, whereby the cell forms colonies with three-dimensional shape.

On the other hand, primed cells have the characteristics of post-implantation embryos and rarely contribute to chimeras and germ line. In brief, primed cells are already at a more differentiated stage compared to nave cells. Primate ES/iPS cells were conventionally believed to be established in primed state6,7. However, recent publications have demonstrated a reliable method for transforming human ES cells from primed to nave state8,9. Transcriptome analysis using RNA-Seq played an important role in identifying the cellular characteristics reported in those articles.

In the case of pig iPS cells, the status of the cellsnave or primedremains inconclusive since the pluripotent genes have a wide variety of phenotypes. To understand the biological variety of pig iPS cells, multiple datasets of global gene expression profiling would be needed. Although a significant number of reports on the establishment of pig iPS cells have been published10,11,12,13,14,15,16,17,18,19,20, expression profiling data in Sequence Read Archive (SRA) database are quite limited. Therefore, detailed biological features of pig iPS cells need to be addressed with whole expression profiling.

In our previous publication, we had reported that pig iPS cell, derived from six reprogramming factors, has more advantageous than that derived from four factors. Especially, the expression of six reprogramming factors was suitable for X chromosome re-activation21, which is one of the mile-stone characteristics of nave-type stem cells. Our previous data using Ion Torrent sequencing also proved that the expression of six reprogramming factors was more advantageous to activate various pluripotent genes. Although the data obtained from Ion Torrent is suggestive, at least 20M reads would be necessary to obtain a quantitative evaluation of the relatively low-expressing genes. The data obtained in our previous publication seem insufficient in terms of the number of sequencing reads required to conclude. This situation led us to detect the global expression profile of pig iPS cells, derived from the expression of six reprogramming factors, using Illumina short-read sequencer, HiSeq 2500. Currently, there are no publicly available dataset of six factor-derived pig iPS cells using Illumina sequencing platform.

The aim of this study was to clarify the difference of mRNA expression profiles between pig iPS cells derived from six and four reprogramming factors. We found relevant submitted data from two research groups on pig iPS cells with four reprogramming factors, in SRA22,23. We could compare ours with these gene expressions since both datasets were obtained with Illumina sequencer. In this study, we describe the detailed expression profile of pig iPS cells derived from four and six reprogramming factors. Multiple analyses demonstrated that the pig iPS cells derived from six factors formed independent clusters compared to those derived from four factors, and were distant from fibroblasts. Furthermore, we detected that the expression levels of various nave-specific genes were relatively elevated in pig iPS cells derived from six factors. Our data set would contribute to the understanding of biological differences between the iPS cells derived from six and four reprogramming factors, and provide the scientific explanation of how diversity of pluripotency-related genes related to the process of animal evolution.

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Global transcriptome analysis of pig induced pluripotent ...