Archive for the ‘Induced Pluripotent Stem Cells’ Category

Induced Pluripotent Stem Cell Market to Reach US$ 2,299.5 …

Induced Pluripotent Stem Cells | Posted by admin
Sep 29 2018

NEW YORK, May 31, 2018 /PRNewswire/

Ongoing Research to Make iPS Cell a Breakthrough Technology for Clinical Research

The healthcare industry has been focusing on excessive research and development in the last couple of decades to ensure that the need to address issues related to the availability of drugs and treatments for certain chronic diseases is effectively met. Healthcare researchers and scientists at the Li Ka Shing Faculty of Medicine of the Hong Kong University have successfully demonstrated the utilization of human induced pluripotent stem cells or hiPSCs from the skin cells of the patient for testing therapeutic drugs.

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The success of this research suggests that scientists have crossed one more hurdle towards using stem cells in precision medicine for the treatment of patients suffering from sporadic hereditary diseases. iPSCs are the new generation approach towards the prevention and treatment of diseases that takes into account patients on an individual basis considering their genetic makeup, lifestyle, and environment. Along with the capacity to transform into different body cell types and same genetic composition of the donors, hiPSCs have surfaced as a promising cell source to screen and test drugs.

Induced Pluripotent Stem Cell Market Research Report Overview @ https://www.persistencemarketresearch.com/market-research/induced-pluripotent-stem-cells-market.asp

In the present research, hiPSC was synthesized from patients suffering from a rare form of hereditary cardiomyopathy owing to the mutations in Lamin A/C related cardiomyopathy in their distinct families. The affected individuals suffer from sudden death, stroke, and heart failure at a very young age. As on date, there is no exact treatment available for this condition.

This team in Hong Kong tested a drug named PTC124 to suppress specific genetic mutations in other genetic diseases into the iPSC transformed heart muscle cells. While this technology is being considered as a breakthrough in clinical stem cell research, the team at Hong Kong University is collaborating with drug companies regarding its clinical application.

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The unique properties of iPS cells provides extensive potential to several biopharmaceutical applications. iPSCs are also used in toxicology testing, high throughput, disease modeling, and target identification. This type of stem cell has the potential to transform drug discovery by offering physiologically relevant cells for tool discovery, compound identification, and target validation. A new report by Persistence Market Research (PMR) states that the global induced pluripotent stem or iPS cell market is expected to witness a strong CAGR of 7.0% from 2018 to 2026. In 2017, the market was worth US$ 1,254.0 Mn and is expected to reach US$ 2,299.5 Mn by the end of the forecast period in 2026.

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Customization to be the Key Focus of Market Players

Due to the evolving needs of the research community, the demand for specialized cell lines have increased to a certain point where most vendors offering these products cannot depend solely on sales from catalog products. The quality of the products and lead time can determine the choices while requesting custom solutions at the same time. Companies usually focus on establishing a strong distribution network for enabling products to reach customers from the manufacturing units in a short time period.

Entry of Multiple Small Players to be Witnessed in the Coming Years

Several leading players have their presence in the global market; however, many specialized products and services are provided by small and regional vendors. By targeting their marketing strategies towards research institutes and small biotechnology companies, these new players have swiftly established their presence in the market.

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Induced Pluripotent Stem Cell Market to Reach US$ 2,299.5 ...

Use Of Induced Pluripotent Stem Cell Models To Elucidate …

Induced Pluripotent Stem Cells | Posted by admin
Sep 29 2018

Degree Name

Doctor of Philosophy (PhD)

Cell & Molecular Biology

Jean Bennett

Choroideremia (CHM) is a rare monogenic, X-linked recessive inherited retinal degenerative disease caused by mutations in the Rab Escort Protein-1 (REP1) encoding CHM gene. CHM is characterized by childhood-onset night blindness (nyctalopia), progressive peripheral vision loss due to the degeneration of neural retina, RPE and choroid in a peripheral-to-central fashion. Most of CHM mutations are loss-of-function mutations leading to the complete lacking of REP1 protein. However, the primary retinal cell type leading to CHM and molecular mechanism remains unknown in addition to the fact of lacking proper disease models. In this study, we explored the utility of induced pluripotent stem cell-derived models of retinal pigment epithelium (iPSC-RPE) to study disease pathogenesis and a potential gene-based intervention in four different genetically distinct forms of CHM. A number of abnormal cell biologic, biochemical, and physiologic functions were identified in the CHM patient cells. Transduction efficiency testing using 11 recombinant adeno-associated virus (AAV) serotype 1-9, 7m8 and 8b showed a differential cell tropism on iPSC and iPSC-derived RPE. We identified AAV7m8 to be optimal for both delivering transgenes to iPSC-RPEs as well as to appropriate target cells (RPE cells and rod photoreceptors) in the primate retina. To establish the proof of concept of AAV7m8 mediated CHM gene therapy, we developed a AAV7m8.hCHM viral vector, which delivers the human CHM cDNA under control of CMV-enhanced chicken -actin promoter (CA). Delivery of AAV7m8.CMV.CA.hCHM to CHM iPSC-RPEs restored protein prenylation, trafficking and phagocytosis defects. The results confirm that AAV-mediated delivery of the REP1-encoding gene can rescue defects in CHM iPSC-RPE regardless of the type of disease-causing mutation. The results also extend our understanding of mechanisms involved in the pathophysiology of choroideremia.

Duong, Thu Thi, "Use Of Induced Pluripotent Stem Cell Models To Elucidate Retinal Disease Pathogenesis And To Develop Gene-Based Therapies" (2018). Publicly Accessible Penn Dissertations. 3003. https://repository.upenn.edu/edissertations/3003

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What Are Induced Pluripotent Cells? – Stem Cell Centers …

Induced Pluripotent Stem Cells | Posted by admin
Aug 23 2018

In 2006, researchers at Kyoto University in Japan established conditions that resulted in specialized adult cells that could be genetically reprogrammed to assume a stem cell-like state. These adult cells, called induced pluripotent stem cells (iPSCs), were successfully reprogrammed to an embryonic stem cell-like state. This was achieved by introducing genes important for maintaining the essential properties of embryonic stem cells (ESCs). Since then, scientists have greatly improved the techniques to engineer iPSCs, creating a powerful new way to de-differentiate cells. iPSCs give scientists an alternative, pluripotent cell to human embryonic which could help with some of the ethical concerns surrounding ESCs.

Induced pluripotent cells provide scientists and doctors tools for drug development, modeling disease, and improving transplantation medicine. Induced pluripotent cells also offer potential resources for cell-replacement therapies and regenerative medicine. One of the challenges with stem cell therapy progress has been with immune rejection the patients body attacks the injected stem cells because it doesnt view them as belonging there. But, with induced pluripotent cells, the source cells are derived from the patient so immune rejection would be much less common.

A field that has greatly prospered since the introduction of iPS cell technology is that of drug testing and development. Scientists can buy different human cells types derived from human iPS cells to test the efficacy or toxicity of new drugs. In the past, scientists used engineered cell lines or rats/mice to model human disease. The opportunity for scientists to use human iPS cells to study human diseases in corresponding human cell types has helped boost the efficacy and process of drug discovery.

The ability to reprogram cell types opens doors for treating numerous diseases including: Parkinsons disease, diabetes, cardiovascular disease, Alzheimers disease and others. In the case of Alzheimers disease, scientists can take a patients skin or blood cells who is afflicted with Alzheimers, and reprogram the cells to produce iPS cells. Then, these iPS cells can be differentiated into numerous cell types found in our brains. These differentiated cells can provide information about what is different between these cells compared to someone who is not afflicted with Alzheimers disease. Understanding the disease better is one of the biggest steps forward to finding effective treatments and prevention methods.

Many diseases stem from genetic defects. Scientists are working to understand the link between disease and genotype. CRISPR is a gene editing technology which helps with understanding this link better. CRISPR is an acronym forClustered Regularly Interspaced Short Palindromic Repeat. The name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms (Harvard, 2014). With CRISPR, a genetic defect from a patient-derived iPS cell can be corrected and then correlated with the original to assist scientists with identifying which genetic elements trigger disease progression.

Despite some of the setbacks and slow-downs of induced pluripotent cell science, the October issue of The Scientist stated that stem-cell based regenerative medicine is getting closer to clinical application. Scientists around the world are employing pluripotent cells to create various therapeutic cell types for diabetes and Parkinsons disease. In 2010, the Geron Corporation began the first FDA-approved clinical trial using human ESCs to treat spinal cord injury. Costs associated with clinical trials continues to be an obstacle for both patients and scientists. The Astellas Institute for Regenerative Medicine (formerly Advanced Cell Technology) is pursuing an embryonic stem cell treatment for macular degeneration, and launched a Phase 2 clinical trial last year.

The May 2017 edition of Science Daily reported that researchers have learned more about how stem cells develop into organs. These scientists were able to grow and purify the earliest lung progenitors that emerge from human stem cells, and then differentiate these cells into tiny bronchospheres that model cystic fibrosis. Scientists are hopeful that these findings will reveal, personalized medicine methods for treating lung disease.

Since their introduction in 2006, induced pluripotent stem cells have created quite a stir. Although their potential has yet to be realized, in time, they will take medicine places that are exciting and promising for those who suffer from disease and life-altering conditions.

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What Are Induced Pluripotent Cells? - Stem Cell Centers ...

Induced Pluripotent Stem Cells (iPSCs)

Induced Pluripotent Stem Cells | Posted by admin
Aug 19 2018

Initially described in the pioneering work of Yamanaka and colleagues, the ability to "reprogram" differentiated somatic cells into a pluripotent embryonic stem cell-like state by retroviral mediated expression of four specific transcription factors has revolutionized our ability to develop new models to study human disease and represents a significant step towards patient-specific cell replacement therapies.

In addition to solving ethical concerns related to the use of blastocyst-derived embryonic stem cells, the use of iPSCs for the generation of therapeutic cells for cell replacement therapy may avoid the requirement for post-transplant immune suppression because iPSCs can be generated directly from the transplant recipient and will therefore be genetically identical to the patient. Additionally, because it is possible to reprogram somatic cells derived from diseased individuals iPSC technology provides an important new platform for the development of new models of human disease. Thus, upon appropriate differentiation these cells can then be used to study normal and pathologic human tissue development in vitro, enabling new insights into disease pathology as well as a platform for the development of novel therapeutic agents and patient-specific cell replacement therapies.

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Induced Pluripotent Stem Cells (iPSCs)

Human Induced Pluripotent Stem Cells – Cell Applications

Induced Pluripotent Stem Cells | Posted by admin
Aug 19 2018

Integration-free HiPSC.Our HiPSC are generated with the RNA-based Sendai virus to deliver reprogramming factors to donor skin fibroblasts. Since the virus does not go through a DNA phase, its genetic material and transgenes do not integrate into the host cell genome. HiPSC are validated for viability, karyotype, pluripotency, plating efficiency, morphology, passage number and lack of contamination.

HiPSC-derived Neural Stem Cells (L) and Neurons (R). i-HNSC stained w/ Nestin (neural stem cell marker, green), SOX 2 (stem cell marker, red) & DAPI (nuclear stain, blue). SOX2 & DAPI nuclear co-localization yields purple (Left). Video: Human iPSC-Derived Neurons establish mature, synchronized neuronal network. In real time, Multi-electrode array (MEA) shows optimal electrophysiological activity, which can be modulated by neurotransmitters or small compounds (Right).

HiPSC-derived Cardiomyocytes (i-HCM) plated onto a flat culture surface pulsate in vitro (L), while iHCM printed into 3D heart tissue using a Cyfuse Regenova also beat (R)

Reprogrammingtriggers a cascade of evident changes in the host cells that are recognizable morphologically and through a combination of markers and pluripotency assays. Our HiPSCs display classic pluripotent stem cell morphology, with a high nucleus to cytoplasm size ratio, as well as they are amenable to be cultivated in serum-free media, independent of feeder cells and of feeder-conditioned media as colonies or high density monolayers. hiPSCs are also evaluated for the presence of karyotypic abnormalities. Confirmation of pluripotency is performed through the analysis of expression of several established independent pluripotency markers. Unguided differentiation confirms HiPSC ability to generate cell derivatives of tissues arising from the three embryonic layers.

Cell Characterization. Post-thawing viability of HiPSCs is typically higher than 70%, and HiPSC have demonstrated coherent pluripotent behavior over more than 60 passages. Although it is in theory possible to propagate HiPSCs indefinitely, HiPSC subculturing over passages higher than is usually not recommended, as the chances of karyotypic abnormalities increase. HiPSCs are also tested to ensure absence of microorganism contaminants.

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Human Induced Pluripotent Stem Cells - Cell Applications

Induced pluripotent stem cell | biology | Britannica.com

Induced Pluripotent Stem Cells | Posted by admin
Jul 04 2018

Alternative Title: iPS cell

Induced pluripotent stem cell (iPS cell), immature cell that is generated from an adult (mature) cell and that has regained the capacity to differentiate into any type of cell in the body. Induced pluripotent stem cells (iPS cells) differ from embryonic stem cells (ES cells), which form the inner cell mass of an embryo but also are pluripotent, eventually giving rise to all the cell types that make up the body. Induced pluripotent cells were first described in 2006 by Japanese physician and researcher Shinya Yamanaka and colleagues. The first experiments were performed by using mouse cells. The following year, however, Yamanaka successfully derived iPS cells from human adult fibroblast cells. Until that time, human stem cells could be obtained only by isolating them from early human embryos. Hence, an important feature of iPS cells is that their generation does not require an embryo, the use of which is fraught with ethical issues.

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stem cell: Induced pluripotent stem cells

Due to the ethical and moral issues surrounding the use of embryonic stem cells, scientists have searched for ways to reprogram adult somatic cells. Studies of cell fusion, in which differentiated adult somatic cells grown in culture with embryonic stem cells

The generation of iPS cells from somatic cells (fully differentiated adult cells, excluding germ cells) was based on the idea that any cell in the body can be reprogrammed to a more primitive (stemlike) state. Among the first to discover that possibility was British developmental biologist John B. Gurdon, who in the late 1950s had shown in frogs that egg cells are able to reprogram differentiated cell nuclei. Gurdon used a technique known as somatic cell nuclear transfer (SCNT), in which the nucleus of a somatic cell is transferred into the cytoplasm of an enucleated egg (an egg that has had its nucleus removed). In 1996 British developmental biologist Ian Wilmut and colleagues used SCNT to create Dolly the sheep, the first clone of an adult mammal. The experiments with SCNT were crucial to the eventual production of iPS cells. Indeed, by the time of Dollys creation, it was widely accepted that factors in the egg cytoplasm were responsible for reprogramming differentiated cell nuclei. The factors controlling the process were unknown, however, until Yamanaka published his first report describing iPS cell generation. (Yamanaka and Gurdon shared the 2012 Nobel Prize for Physiology or Medicine for their discoveries.)

Several proteins have been identified that are capable of inducing or enhancing pluripotency in nonpluripotent (i.e., adult) cells. Of key importance are the transcription factors Oct-4 (octamer 4) and Sox-2 (sex-determining region Y box 2), which maintain stem cells in a primitive state. Other proteins that may be used to enhance pluripotency include Klf-4 (Kruppel-like factor 4), Nanog, and Glis1 (Glis family zinc finger 1).

Pluripotency factors can be introduced into nonpluripotent cells in different ways, such as by plasmids or delivery as proteins or modified RNAs. Among the most effective and widely used methods, however, is delivery via a retroviral vector. Retroviral vectors can readily enter cells, making the genes they carry accessible to the cell; other retroviral activities are silenced. However, because retroviruses integrate into the nuclear genome, their use raises the risk of virus-induced tumour formation. Nonetheless, retroviral delivery remains highly effective, and technical advances to prevent the integration of retroviral material into the nuclear genome have allowed for the generation of iPS cells via ectopic expression (in the cytoplasm) of retrovirus-delivered transcription factors. Ectopic expression also has been achieved with the use of recombinant adeno-associated virus.

Since the initial development of iPS cells, researchers have been working to improve the techniques and to learn what drives pluripotent stem cells to differentiate in particular ways. They also have been investigating the use of iPS cells in the treatment of certain diseases. Of significance is the potential to create patient-specific iPS cells (using a patients own adult cells), which could allow for the generation of perfectly matched cells and tissues for transplantation therapies. Such therapies could help overcome the risk of immune rejection, which is a major challenge in regenerative medicine.

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Induced Pluripotent Stem Cell FAQs | Sigma-Aldrich

Induced Pluripotent Stem Cells | Posted by admin
Jul 04 2018

What are induced pluripotent stem cells (iPSCs)? Induced pluripotent stem cells (iPSCs or iPS cells) are a type of pluripotent stem cell that can be generated from adult somatic cells such as skin fibrobalsts or peripheral blood mononuclear cells (PBMCs) by genetic reprograming or the 'forced' introduction of reprogramming genes (Oct4, Sox2, Klf4 and c-Myc).

How are the induced pluripotent stem (iPS) cells produced? In 2006, Shinya Yamanaka produced the first iPS cells - murine ES (embryonic stem) like cell lines - from mouse embryonic fibroblasts (MEFs) and skin fibroblasts by inserting four transcription factor genes encoding Oct4, Sox2, Klf4, and c-Myc. Another group of researchers identified two other genes, Nanog, and Lin28 as a replacement of Klf4, and c-Myc to reprogram human cells. The source of reprogramming genes could be generated from various origins, including neuronal progenitor cells, keratinocytes, hepatocytes, B cells, and fibroblasts of mouse-tail tips, kidneys, muscles, and adrenal glands. Fusion of two types of cells could convert specialized cell types from one lineage to another. These newly developed cells possess similar morphology and growth characteristics as parent ES cells by expressing ES cell-specific genes. The success of reprogramming iPS cell technology depends on the sources of cell lines. It has been reported that reprogramming of human keratinocyte cells withdrawn from skin biopsies to pluripotency proceed at much higher frequency and faster speed than fibroblasts. Recently, non-integrating methods of reprogramming have become popular including RNA sendai virus and RNA based reprogramming methods.

What are the advantages of iPS cells over embryonic stem cells? The advantage of iPS cells is that they are not derived from human embryos, which is the ethical concern in this field. By removing the bioethical issues, the scientists are more likely to obtain more federal funding and support. Another significant benefit of iPS cell technology would permit for creation of isogenic control cell lines using CRISPR/Cas9 gene editing that are genetically tailored to model a disease phenotype.

What are the risks associated with iPS cell use in humans? The retroviruses used in the generation of iPSCs are associated with cancer because they insert DNA anywhere in a cell's genome, which could potentially trigger the expression of cancer-causing genes. Another risk associated with iPS cell technology applied to humans is the fact that c-Myc, which is one of the genes used in reprogramming, is a known oncogene whose overexpression could also cause cancer. In addition, the successful reprogramming rate in human iPS cells from fibroblasts is fairly low (

How are iPS cells similar to ES cells? iPS cells are similar to ES cells in morphology, teratoma formation, proliferation, expression of pluripotency markers, long telomeric zone, generation of embryoid bodies and viable chimeras as well as their ability to differentiate along a given lineage. They also express stem cell surface markers and genes that characterize ES cells such as Oct4, Sox2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4 and Nanog.

Does iPS cell technology eliminate the need for embryonic stem cell research? Recent advances do not eliminate the need for ES cell research since it is not yet quite clear whether iPS cells differ extensively from the embryonic stem cells. To bring stem cell research to clinical realization, it is necessary to investigate all the aspects in this field such as the most efficient stem cell for cell replacement therapies.

What are disease specific iPS cells? Disease specific iPS cells are iPS cells generated from subjects with a genetic disease. These cells, generated from patients with untreatable diseases, can be used to study the pathophysiology of various diseases in vitro and enable drug development. Another significant benefit of iPS cell technology would permit for creation of isogenic control cell lines using CRISPR/Cas9 gene editing that are genetically tailored to a patient or disease phenotype.

Where can obtain human iPS cells? The European Bank of Induced Pluripotent Stem Cells (EBiSC) is a collection of high quality human iPS cells available for researchers for use in disease modelling and other forms of stem cell research. The initial collection has been generated from a wide range of donors representing specific disease backgrounds and healthy controls. EBiSC depositors have established many routine procedures for collecting, expanding and characterizing human iPS cell lines. The stem cell bank includes iPSC cell lines derived from neurodegenerative diseases (Alzheimers Disease, Parkinsons Disease, Dementia, Motor Neuron Disease (ALS) - and Huntingtons Disease), eye and heart diseases, and lines from healthy control donors for age and sex matching.

How are iPS cells grown in culture? The ability to expand human iPSCs in vitro and subject them to cell-type specific differentiation protocols is critical for generating patient derived disease-in-a-dish cellular models for basic stem cell research and drug-discovery applications. Standardized iPSC protocols on how to thaw, culture and cryopreserve human induced pluripotent stem cells (iPSCs) have been established by the European Bank of induced pluripotent Stem Cells (EBiSC). Human induced pluripotent stem cell (iPSC) lines are different to any other established cell line. If you are not familiar with culturing iPSCs make sure you read the following instructions carefully. Recently, 3D cell culture organoid models have utilized iPS cells to more accurately model many organ systems in vitro.

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Induced Pluripotent Stem Cell FAQs | Sigma-Aldrich

Induced Pluripotent Stem Cell India – StemCellCareIndia

Induced Pluripotent Stem Cells | Posted by admin
Jul 02 2018

Induced pluripotent stem cells (iPSCs) area unit adult cells that are genetically reprogrammed to AN embryonic stem celllike state by being forced to specific genes and factors necessary for maintaining the shaping properties of embryonic stem cells. though these cells meet the shaping criteria for pluripotent stem cells, its not well-known if iPSCs and embryonic stem cells take issue in clinically vital ways in which. Mouse iPSCs were 1st according in 2006, and human iPSCs were 1st according in late 2007. Mouse iPSCs demonstrate necessary characteristics of pluripotent stem cells, together with expressing somatic cell markers, forming tumors containing cells from all 3 germ layers, and having the ability to contribute to several completely different tissues once injected into mouse embryos at a really early stage in development. Human iPSCs additionally specific somatic cell markers and area unit capable of generating cells characteristic of all 3 germ layers.

Although further analysis is required, iPSCs area unit already helpful tools for drug development and modeling of diseases, and scientists hope to use them in transplantation medication. Viruses area unit presently wont to introduce the reprogramming factors into adult cells, and this method should be rigorously controlled and tested before the technique will cause helpful treatment for humans. In animal studies, the virus wont to introduce the somatic cell factors generally causes cancers. Researchers area unit presently work non-viral delivery ways. In any case, this breakthrough discovery has created a robust new thanks to de-differentiate cells whose organic process fates had been antecedently assumed to be determined. additionally, tissues derived from iPSCs are an almost identical match to the cell donor and so in all probability avoid rejection by the system. The iPSC strategy creates pluripotent stem cells that, in conjunction with studies of different varieties of pluripotent stem cells, can facilitate researchers learn the way to reprogram cells to repair broken tissues within the figure.

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Cell potency – Wikipedia

Induced Pluripotent Stem Cells | Posted by admin
Jun 27 2018

Cell potency is a cell's ability to differentiate into other cell types[1][2][3] The more cell types a cell can differentiate into, the greater its potency. Potency is also described as the gene activation potential within a cell which like a continuum begins with totipotency to designate a cell with the most differentiation potential, pluripotency, multipotency, oligopotency and finally unipotency.

Totipotency (Lat. totipotentia, "ability for all [things]") is the ability of a single cell to divide and produce all of the differentiated cells in an organism. Spores and zygotes are examples of totipotent cells.[4] In the spectrum of cell potency, totipotency is a form of pluripotency that represents the cell with the greatest differentiation potential.

It is possible for a fully differentiated cell to return to a state of totipotency.[5] This conversion to totipotency is complex, not fully understood and the subject of recent research. Research in 2011 has shown that cells may differentiate not into a fully totipotent cell, but instead into a "complex cellular variation" of totipotency.[6] Stem cells resembling totipotent blastomeres from 2-cell stage embryos can arise spontaneously in mouse embryonic stem cell cultures[7][8] and also can be induced to arise more frequently in vitro through down-regulation of the chromatin assembly activity of CAF-1.[9]

The human development model is one which can be used to describe how totipotent cells arise.[10] Human development begins when a sperm fertilizes an egg and the resulting fertilized egg creates a single totipotent cell, a zygote.[11] In the first hours after fertilization, this zygote divides into identical totipotent cells, which can later develop into any of the three germ layers of a human (endoderm, mesoderm, or ectoderm), or into cells of the placenta (cytotrophoblast or syncytiotrophoblast). After reaching a 16-cell stage, the totipotent cells of the morula differentiate into cells that will eventually become either the blastocyst's Inner cell mass or the outer trophoblasts. Approximately four days after fertilization and after several cycles of cell division, these totipotent cells begin to specialize. The inner cell mass, the source of embryonic stem cells, becomes pluripotent.

Research on Caenorhabditis elegans suggests that multiple mechanisms including RNA regulation may play a role in maintaining totipotency at different stages of development in some species.[12] Work with zebrafish and mammals suggest a further interplay between miRNA and RNA-binding proteins (RBPs) in determining development differences.[13]

In cell biology, pluripotency (Lat. pluripotentia, "ability for many [things]")[14] refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system).[15] However, cell pluripotency is a continuum, ranging from the completely pluripotent (or totipotent) cell that can form every cell of the embryo proper, e.g., embryonic stem cells and iPSCs (see below), to the incompletely or partially pluripotent cell that can form cells of all three germ layers but that may not exhibit all the characteristics of completely pluripotent cells.

Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs, are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of certain genes and transcription factors.[16] These transcription factors play a key role in determining the state of these cells and also highlights the fact that these somatic cells do preserve the same genetic information as early embryonic cells.[17] The ability to induce cells into a pluripotent state was initially pioneered in 2006 using mouse fibroblasts and four transcription factors, Oct4, Sox2, Klf4 and c-Myc;[18] this technique, called reprogramming, earned Shinya Yamanaka and John Gurdon the Nobel Prize in Physiology or Medicine 2012.[19] This was then followed in 2007 by the successful induction of human iPSCs derived from human dermal fibroblasts using methods similar to those used for the induction of mouse cells.[20] These induced cells exhibit similar traits to those of embryonic stem cells (ESCs) but do not require the use of embryos. Some of the similarities between ESCs and iPSCs include pluripotency, morphology, self-renewal ability, a trait that implies that they can divide and replicate indefinitely, and gene expression.[21]

Epigenetic factors are also thought to be involved in the actual reprogramming of somatic cells in order to induce pluripotency. It has been theorized that certain epigenetic factors might actually work to clear the original somatic epigenetic marks in order to acquire the new epigenetic marks that are part of achieving a pluripotent state. Chromatin is also reorganized in iPSCs and becomes like that found in ESCs in that it is less condensed and therefore more accessible. Euchromatin modifications are also common which is also consistent with the state of euchromatin found in ESCs.[21]

Due to their great similarity to ESCs, iPSCs have been of great interest to the medical and research community. iPSCs could potentially have the same therapeutic implications and applications as ESCs but without the controversial use of embryos in the process, a topic of great bioethical debate. In fact, the induced pluripotency of somatic cells into undifferentiated iPS cells was originally hailed as the end of the controversial use of embryonic stem cells. However, iPSCs were found to be potentially tumorigenic, and, despite advances,[16] were never approved for clinical stage research in the United States. Setbacks such as low replication rates and early senescence have also been encountered when making iPSCs,[22] hindering their use as ESCs replacements.

Additionally, it has been determined that the somatic expression of combined transcription factors can directly induce other defined somatic cell fates (transdifferentiation); researchers identified three neural-lineage-specific transcription factors that could directly convert mouse fibroblasts (skin cells) into fully functional neurons.[23] This result challenges the terminal nature of cellular differentiation and the integrity of lineage commitment; and implies that with the proper tools, all cells are totipotent and may form all kinds of tissue.

Some of the possible medical and therapeutic uses for iPSCs derived from patients include their use in cell and tissue transplants without the risk of rejection that is commonly encountered. iPSCs can potentially replace animal models unsuitable as well as in vitro models used for disease research.[24]

Recent findings with respect to epiblasts before and after implantation have produced proposals for classifying pluripotency into two distinct phases: "naive" and "primed".[25] The baseline stem cells commonly used in science that are referred as Embryonic stem cells (ESCs) are derived from a pre-implantation epiblast; such epiblast is able to generate the entire fetus, and one epiblast cell is able to contribute to all cell lineages if injected into another blastocyst. On the other hand, several marked differences can be observed between the pre- and post-implantation epiblasts, such as their difference in morphology, in which the epiblast after implantation changes its morphology into a cup-like shape called the "egg cylinder" as well as chromosomal alteration in which one of the X-chromosomes undergoes random inactivation in the early stage of the egg cylinder, known as X-inactivation.[26] During this development, the egg cylinder epiblast cells are systematically targeted by Fibroblast growth factors, Wnt signaling, and other inductive factors via the surrounding yolk sac and the trophoblast tissue,[27] such that they become instructively specific according to the spatial organization.[28] Another major difference that was observed, with respect to cell potency, is that post-implantation epiblast stem cells are unable to contribute to blastocyst chimeras,[29] which distinguishes them from other known pluripotent stem cells. Cell lines derived from such post-implantation epiblasts are referred to as epiblast-derived stem cells which were first derived in laboratory in 2007; despite their nomenclature, that both ESCs and EpiSCs are derived from epiblasts, just at difference phases of development, and that pluripotency is still intact in the post-implantation epiblast, as demonstrated by the conserved expression of Nanog, Fut4, and Oct-4 in EpiSCs,[30] until somitogenesis and can be reversed midway through induced expression of Oct-4.[31]

Multipotency describes progenitor cells which have the gene activation potential to differentiate into discrete cell types. For example, a multipotent blood stem cell and this cell type can differentiate itself into several types of blood cell types like lymphocytes, monocytes, neutrophils, etc., but it is still ambiguous whether HSC possess the ability to differente into brain cells, bone cells or other non-blood cell types.[citation needed]

New research related to multipotent cells suggests that multipotent cells may be capable of conversion into unrelated cell types. In another case, human umbilical cord blood stem cells were converted into human neurons.[32] Research is also focusing on converting multipotent cells into pluripotent cells.[33]

Multipotent cells are found in many, but not all human cell types. Multipotent cells have been found in cord blood,[34] adipose tissue,[35] cardiac cells,[36] bone marrow, and mesenchymal stem cells (MSCs) which are found in the third molar.[37]

MSCs may prove to be a valuable source for stem cells from molars at 810 years of age, before adult dental calcification. MSCs can differentiate into osteoblasts, chondrocytes, and adipocytes.[38]

In biology, oligopotency is the ability of progenitor cells to differentiate into a few cell types. It is a degree of potency. Examples of oligopotent stem cells are the lymphoid or myeloid stem cells.[2] A lymphoid cell specifically, can give rise to various blood cells such as B and T cells, however, not to a different blood cell type like a red blood cell.[39] Examples of progenitor cells are vascular stem cells that have the capacity to become both endothelial or smooth muscle cells.

In cell biology, a unipotent cell is the concept that one stem cell has the capacity to differentiate into only one cell type. It is currently unclear if true unipotent stem cells exist. Hepatoblasts, which differentiate into hepatocytes (which constitute most of the liver) or cholangiocytes (epithelial cells of the bile duct), are bipotent.[40] A close synonym for unipotent cell is precursor cell.

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Cell potency - Wikipedia

Induced Pluripotent Stem Cells | The Progeria Research Foundation

Induced Pluripotent Stem Cells | Posted by admin
Oct 14 2017

The Progeria Research Foundation Cell & Tissue BankHuman Induced Pluripotent Stem Cells (iPSC)

1. iPSC Background information for the non-scientist Stem cells are immature cells that have not yet committed to becoming any one cell type. They are pliable because they have the potential to develop into many different types of mature cells in the body, such as cells that make up the heart or blood vessels, and other tissues and organs. In 2007, researchers discovered a strategy for creating stem cells in the laboratory by reprogramming mature adult cells that we commonly grow for research purposes.1, 2 . These artificially created stem cells are called Induced Pluripotent Stem Cells (iPSCs). For the field of Progeria, this is a huge breakthrough. For the first time, scientists can now make Progeria stem cells and ask questions about how stem cells function and develop in Progeria. Previously there was no source of human Progeria stem cells, and there was therefore a void of information about how Progeria stem cells function compared with stem cells from people without Progeria. In addition, scientists can re-program the Progeria stem cells to create, for the first time, mature Progeria blood vessels, heart cells, and other cell types. Until now, there was no source of human Progeria heart or blood vessel cells. We can now ask key questions about the heart disease that leads to early death in Progeria from heart attacks and strokes. We can compare these discoveries with the heart disease and aging in the general population and discover more about what influences aging in all of us. Already there have been several excellent studies published using Progeria stem cells.3-5 Our goal at The Progeria Research Foundation is to facilitate many more discoveries using this invaluable tool. For a primer on stem cells, please see this US government website: http://stemcells.nih.gov/info/basics/defaultpage.asp

2.Purpose of induced pluripotent stem cell (iPSC) generation and distribution by The Progeria Research Foundation The mission of The Progeria Research Foundation is to discover treatments and the cure for Hutchinson-Gilford Progeria Syndrome and its aging-related disorders. In 2009, PRF entered into a collaboration with an expert team of scientists at the University of Toronto, Canada, under the direction of William Stanford, PhD, to generate high quality Progeria iPSCs. Dr. Stanford is the Canada Research Chair in Integrative Stem Cell Biology. As of 2011, PRF continues to collaborate with Dr. Stanford at the University of Ottawa, Canada where he is Professor of Cellular and Molecular Medicine, Faculty of Medicine, and Senior Scientist at Ottawa Hospital Research Institutes Sprott Centre for Stem Cell Research.

Our goal is to provide this invaluable tool to researchers throughout the world. This new research tool will be used to generate new and innovative research in Progeria, as well as its relationship to heart disease and aging.

3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs) Induced-Pluripotent Stem Cells (iPSCs) were derived using VSVG-pseudotyped retroviral transduction of four human factors, Oct4, Sox2, Klf4, and c-Myc into fibroblasts. iPSC colonies were derived on mouse-embryonic fibroblasts (MEFs). The procedure used was essentially as previously described but without the use of the EOS reporter (Nature Protocols 4: 1828-1844, 2009).

4. Quality Control: Validation and Characterization The lines that are currently available have undergone several validation steps (see downloadable PDFs below):

Additional validation in process: Some lines have completed teratoma assays as shown in supporting data. For all other lines, teratoma assays are in process and status will be updated as these assays are completed.

5. Original starting material from which these iPS cells were derived iPSCs were derived from PRF Cell & Tissue Bank non-transformed fibroblast cell lines.

The transduction method used for all iPS lines was Retrovirus MKOS.

iPSC Line ID

Mutation

Gender and Donation Age

HGADFN003 iPS 1B

LMNAExon 11, 1824 C>T

Male 2yr 0mo

Dermal Fibroblasts HGADFN003

HGADFN003 iPS 1C

LMNA Exon 11, 1824 C>T

Male 2yr 0mo

Dermal Fibroblasts HGADFN003

HGDFN003 iPS 1D

LMNA Exon 11, 1824 C>T

Male 2yr 0mo

Dermal Fibroblasts HGADFN003

HGADFN167 iPS 1J

LMNA Exon 11, 1824 C>T

Male 8yr 5mo

Dermal Fibroblasts HGADFN167

HGADFN167 iPS 1Q

LMNA Exon 11, 1824 C>T

Male 8yr 5mo

Dermal Fibroblasts HGADFN167

HGMDFN090 iPS 1B

Mother of HGADFN167 (unaffected)

Female 37yr 10mo

Dermal Fibroblasts HGMDFN090

HGMDFN090 iPS 1C

Mother of HGADFN167 (unaffected)

Female 37yr 10mo

Dermal Fibroblasts HGMDFN090

HGFDFN168 iPS1 D2

Father of HGADFN167 (unaffected)

Male 40yr 5mo

Dermal Fibroblasts HGFDFN168

HGFDFN168 iPS1P

Father of HGADFN167 (unaffected)

Male 40yr

5mo

Dermal Fibroblasts HGFDFN168

PRF AVAILABLE CELL LINES

6. Join our email list for future iPSC updates and new cell lines We are continuing to generate iPSC lines. If you would like periodic updates on iPSCs held in the PRF Cell & Tissue Bank,please join our emailing list by clicking here

7. Questions? Please contact Leslie Gordon, MD, PhD, Medical Director, with any questions or needs, at lgordon@progeriaresearch.org or 978-535-2594

8. Ordering iPS cell lines

In 2014, PRF instituted a policy of no changes to our MTA. This is the result of 12 years of contractual arrangements with 70 research teams working at institutions in 14 countries. PRF and its counsel have taken into consideration the issues that have arisen in that time period and edited the agreement accordingly, resulting in what we feel are fair and reasonable terms.

For U.S. Federal Government Institutions, please contact Joan Brazier, Research Study Coordinator, at joan_brazier@brown.edu or 401-863-9628.

Step 1: Complete an application and material transfer agreement Application and Agreement for Non-government Institutions

Material Transfer Agreement for Non-government Institutions*

Step 2: Return the completed application and material transfer agreement to PRF at info@progeriaresearch.org. Once approved, you will receive an email confirming your order and anticipated shipping date.

Step 3: Dr. Stanfords laboratory is currently distributing lines as live cultures. His laboratory will email you when the culture has been shipped, with shipping and tracking information. Inexperienced researchers are directed to obtain training at specialized courses essential to human embryonic stem cell/iPSCs work.

Step 4: The University of Ottawa will charge $84.00 per iPSC line plus courier costs, if any, and will send you a bill directly.

9. HGPS and Control iPS Cell Culture Media Preparation Culturing Progeria iPSCs requires the preparation of various kinds of media depending on the growth conditions of the cells and the experimental requirements. In addition to maintenance media, there is also supportive media for the MEFs. The HGPS iPSCs were derived using a Knock-Out medium containing Knock Out Serum Replacement (KOSR).

MEF medium

Store at 4C and use within 4 weeks. If purchasing untreated MEFs from Millipore it is recommended to increase the FBS concentration to 20% for better growth during expansion.

HGPS and Control hiPSC media

Reagent

We recommend Lot testing the Knockout Serum Replacement on established hES cells before being used for Progeria iPS cells.

10. Preparation of HGPS and Control iPSC Culture Surfaces To maintain high quality cells and colonies, it is imperative to passage onto appropriate surfaces. This surface could consist of inactivated mouse embryonic fibroblasts (MEFs, replication arrested through irradiation or mitomycin-c treatment). The protocol for inactivation of MEFs by irradiation follows. However MEFs can also be inactivated by treatment with mitomycin C if there is no access to an irradiator. Inactivated MEFs can be made in house or purchased through Millipore (cat# PMEF-CFL for MEFs that have not been mitotically inactivated or cat# PMEF-CFL for inactivated ones that are ready to use). A vial of untreated MEFs can be expanded and treated with Mitomycin C used immediately or frozen down for future use.

11. Inactivating (by irradiation) and plating MEFs

Materials:

Procedure:

12. Thawing HGPS and Control iPS cell lines One vial of hiPSCs should be thawed into one well of a 6-well plate containing inactivated mouse embryonic feeders cells (MEFs).

Have all tubes, warmed medium, and plates ready before starting the protocol to ensure that the thawing procedure is done as quickly as possible.

Materials:

Procedure:

Note: If only a few undifferentiated colonies are observed after thawing, it may be necessary to select only these colonies for passaging and replate them in the same size well on a new plate.

13. Routine Passaging and Maintenance of Undifferentiated HGPS and Control iPSCs

In order to assure healthy cells, it is important to change their media on a daily basis. This is a simple process of aspirating the old media and replacing it with fresh iPS media. After some time, usually 4-6 days after splitting, it will be necessary to split the cells once again. Splitting cells before they become too confluent will ensure a higher number of undifferentiated cells. Usually a 1:6 or 1:8 split will work well and allow 6-7 days between passages.

14. Suggested Protocol for Passaging iPS CellsUpdated September 4, 2014

The following protocol, obtained from Beers et al, 2012 has been giving excellent results for the team at the Human Pluripotent Stem Cell Facility of Ottawa Hospital Research Institute. According to this team, the protocol has dramatically helped to decrease the differentiated cells that might start to grow and it speeds up the passaging. Cells are often ready within 3 to 5 days instead of 5 to 7. Therefore this could save time and money on media.

EDTA solution: Add 500ul of 0.5M EDTA (pH 8.0) into 500ml of DPBS (-/-). Add 0.9g of NaCl and adjust the osmolarity to 340 mOsm. Filter the solution to sterilize and store it at 4C for up to 6 months. The goal is to create the least amount of disturbance for the cells during dissociation. Therefore the EDTA solution is at the same osmolarity as the E8 media.

Protocol

Add 2ml of E8 media to a 6 well matrigel coated plate.

Take the plate to be passaged and remove the media from the well and wash twice with 1ml of PBS(-/-).

Add 1ml of the EDTA solution to the well and leave for 4min at room temperature.

Once 4 min. is up remove EDTA solution and add 1ml of E8 media.

Scrape cells and divide cells amongst the 6 wells of your plate containing E8 media (Ive been taking 160ul into each well). Avoid breaking up the pieces as much as possible. Preferably use a wide mouth pipette tip.

Swirl and incubate at 37C.

NOTE: Once the cells have been scraped, transfer them to the new plate as soon as possible because the cells will re-attach quickly.

Source:

Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions

Jeanette Beers, Daniel R. Gulbranson, Nicole George, Lauren I. Siniscalchi, Jeffrey Jones, James A. Thomson, and Guokai Chen

Nat Protoc. 2012 Nov; 7(11):2029-40

15. Culturing HGPS and Control iPSCs on MEFs

Materials:

Procedure:

16. Cryopreservation of HGPS and Control iPSCs

Multiple passaging and expansion of iPSCs will result in a surplus of cells. Instead of disposing, it is good practice to freeze cells on occasion to build up a stock and give you cells you can go back to and thaw out for use in the future.

The protocols described below are based on iPSC cultures in 6-well plates where initial clump seeding is adjusted so that wells are 60 70% confluent at time of cryopreservation. Before cryopreservation, iPSCs should be of high quality (primarily undifferentiated with less than 20% of the cells being differentiated). Cryopreservation should be done approximately 1 day before the cells are ready to passage. iPSCs will have improved survival following thawing if cryopreserved as large clumps.

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Induced Pluripotent Stem Cells | The Progeria Research Foundation