Category Archives: Induced Pluripotent Stem Cells

Japanese lab to collaborate with Christian Dior in iPS cell research – Japan Today

An iPS cell research center at one of Japan's top universities said said Thursday that it has entered into a collaborative research project to explore skin rejuvenation with the perfumery and cosmetics division of luxury French fashion house Christian Dior SE.

The Center for iPS Cell Research and Application at Kyoto University, a leading center for induced pluripotent stem cell research, will work with Parfums Christian Dior to analyze what factors are linked to certain signs of aging, such as wrinkles, by comparing the state of skin cells generated from the iPS cells of young and elderly donors.

In the future, the project will also investigate what substances are necessary for regeneration, and how skin cells change after being subjected to various everyday stresses, such as ultraviolet radiation and heat.

Dior Science, the research arm of the luxury brand, has for the past 20 years been exploring how skin cells transform with age and has made a series of discoveries in the cutaneous domain. It aims to utilize the center's stem cell technology to develop ways of regenerating skin cells and maintaining youthful skin.

The collaborative project also hopes to investigate the effects of advancing age on the status of mitochondria, which creates energy for cells, and conduct research using the laboratory's expertise on genome editing.

The center continues to conduct innovative research on the medical applications of iPS cells, which can be converted into any type of cell in the body, including regenerative medicine and the development of new drugs.

Japanese lab to collaborate with Christian Dior in iPS cell research - Japan Today

Induced Pluripotent Stem Cell Market Research Report 2019 From TBRC has Been Updated – Market Research Gazette

A recent report published by The business research Company on Induced Pluripotent Stem Cell (IPSC) Market provides in-depth analysis of segments and sub-segments in the global as well as regional market.

The induced pluripotent stem cell (IPSC) market is a segment of the healthcare services market. The report will answer questions such as where the largest and fastest growing market is, how the market relates to the overall economy, demography and other similar markets, and what forces will shape the market going forward.

The induced pluripotent stem cell (iPSC) market consists of sales of induced pluripotent stem cells and related services. Induced pluripotent stem cells are the regenerated form of stem cells, which are produced from an existing adult cell, such as from hepatocytes, fibroblasts, keratinocytes and neurons.

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Increase in the prevalence of chronic disorders is one of the major factors that is driving the growth of Induced pluripotent stem cell market. Chronic disorders like heart disease, cancer, stroke, diabetes can be treated with Induced pluripotent stem cell. Induced Pluripotent stem cells are taken from any tissues from a child or an adult and are genetically modified to behave like embryonic stem cells.

The potential risk of tumor is one of the major restraints on the growth of Induced pluripotent stem cell market. As per a scientific research, it was found that there might be a chance of getting cancer from the treatment and people are unwilling to take treatment through Induced pluripotent stem cell therapy.

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Induced Pluripotent Stem Cell Market Research Report 2019 From TBRC has Been Updated - Market Research Gazette

What are Induced Pluripotent Stem (IPS) Cells? – Stem Cell …

However, there are a few key differences between these cells. For starters, iPS cells are not dependent on the use of cells from an embryo. Some research also indicates that some of the genes in induced pluripotent cells can behave differently compared to those in embryonic stem cells. This can be caused by an incomplete reprogramming of the cells or genetic changes acquired by the iPS cells as they multiply and grow.

Researchers still have a lot to study in terms of understanding how reprogramming works inside a cell, which is why many scientists believe iPS cells cant replace embryonic stem cells in basic research.

Though iPS cells are a large advancement for medical research, there is still a lot of research to be done in terms of understanding exactly how the reprogramming occurs in a cell. Researchers must also study iPS cells more to understand how iPS cells can be produced consistently and controlled enough to meet quality and safety requirements for the lab/clinic, as well how cells made from iPS cells will behave in the body. iPS cells behave differently from embryonic stem cells, and researchers are still looking at the effects these may have on medicine and research. There is still more to be done in terms of developing affordable and effective iPS cell treatments, as this still remains a constant challenge.

By studying iPS cells closely, scientists were able to develop a way to create these cells without making permanent genetic changes that were linked to the possible formation of tumors. Research and close studies are allowing the creation of iPS obtained specialized cells that will be safe for use for patients, but again, there is still a lot more research to be done.

IPS Cells and The Future

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What are Induced Pluripotent Stem (IPS) Cells? - Stem Cell ...

Induced Pluripotent Stem Cell – ScienceDirect

A.. Owaidah, W.. Kafienah, in Reference Module in Biomedical Sciences, 2016

Induced pluripotent stem cells are believed to be an alternative source for ESC in cell based therapies. These cells were found to share similar characteristics to ESCs in terms of unlimited self-renewal capacity and pluripotency. Their capacity to generate cartilaginous tissue was first evident in teratomas in vivo (Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Boulting et al., 2011; Ellis et al., 2009; Yu et al., 2007). This suggested that iPSC exhibit the capacity to differentiate down the chondrogenic lineage. The methods reported on the derivation of chondrocytes from iPSCs were similar to those reported with ESCs, including techniques based on EB formation, co-culture methods with normal chondrocytes and direct differentiation (Guzzo et al., 2013; Wei et al., 2012; Yamashita et al., 2013; Kim et al., 2011).

Despite the fact that iPSC represent an alternative source to ESC in cell-based therapies, the genomic changes that are associated with most of the reprogramming methods hinder the progression towards clinical applications. In 2014, Borestrom and colleagues (Borestrm et al., 2014) showed that reprograming articular chondrocytes using RNA-based methods eliminates the risk of genomic integration and aberrations. In the same study, reprogrammed cells were successfully differentiated into chondrocytes using two different methods. One protocol involved the directed differentiation on monolayer method presented by Oldershaw et al in 2010. The second method involved the generation of cartilage pellets from iPSC with the supplementation of exogenous TGF-1. Chondrogenic differentiation and cartilage nodule formation were again assessed only through chondrogenic gene expression and alcian blue staining without accurate, quantitative matrix analysis.

In conclusion, although there are various methods that can be used to achieve chondrogenic differentiation of both ESCs and iPSCs, current methods only allow for the generation of small scale chondrogenic cultures without methods for competent scale up that permit large-scale tissue engineering for the repair of bona fide cartilage defects.

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Induced Pluripotent Stem Cell - ScienceDirect

Stem Cell Glossary A Closer Look at Stem Cells

Adult stem cells A commonly used term fortissue-specific stem cells, cells that can give rise to the specialized cells in specific tissues. Includes all stem cells other than pluripotent stem cells such as embryonic and induced pluripotent stem cells.


Autologous Cells or tissues from the same individual; an autologous bone marrow transplant involves one individual as both donor and recipient.


Basic research Research designed to increase knowledge and understanding (as opposed to research designed with the primary goal to solve a problem).


Blastocyst A transient, hollow ball of 150 to 200 cells formed in early embryonic development that contains the inner cell mass, from which the embryo develops, and an outer layer of cell called the trophoblast, which forms the placenta.


Bone marrow stromal cells A general term for non-blood cells in the bone marrow, such as fibroblasts, adipocytes (fat cells) and bone- and cartilage-forming cells that provide support for blood cells. Contained within this population of cells are multipotent bone marrow stromal stem cells that can self-renew and give rise to bone, cartilage, adipocytes and fibroblasts.


Cardiomyocytes The functional muscle cells of the heart that allow it to beat continuously and rhythmically.


Clinical translation The process of using scientific knowledge to design, develop and apply new ways to diagnose, stop or fix what goes wrong in a particular disease or injury; the process by which basic scientific research becomes medicine.


Clinical trial Tests on human subjects designed to evaluate the safety and/or effectiveness of new medical treatments.


Cord blood The blood in the umbilical cord and placenta after child birth. Cord blood contains hematopoietic stem cells, also known as cord blood stem cells, which can regenerate the blood and immune system and can be used to treat some blood disorders such as leukemia or anemia. Cord blood can be stored long-term in blood banks for either public or private use. Also called umbilical cord blood.


Cytoplasm Fluid inside a cell, but outside the nucleus.


Differentiation The process by which cells become increasingly specialized to carry out specific functions in tissues and organs.


Drug discovery The systematic process of discovering new drugs.


Drug screening The process of testing large numbers of potential drug candidates for activity, function and/or toxicity in defined assays.


Embryo Generally used to describe the stage of development between fertilization and the fetal stage; the embryonic stage ends 7-8 weeks after fertilization in humans.


Embryonic stem cells (ESCs) Undifferentiated cells derived from the inner cell mass of the blastocyst; these cells have the potential to give rise to all cell types in the fully formed organism and undergo self-renewal.


Fibroblast A common connective or support cell found within most tissues of the body.


Glucose A simple sugar that cells use for energy.


Hematopoietic Blood-forming; hematopoietic stem cells give rise to all the cell types in the blood.


Immunomodulatory The ability to modify the immune system or an immune response.


Induced pluripotent stem cells (iPSCs)Embryonic-like stem cells that are derived from reprogrammed, adult cells, such as skin cells. Like ESCs, iPS cells are pluripotent and can self-renew.


In vitro Latin for in glass. In biomedical research this refers to experiments that are done outside the body in an artificial environment, such as the study of isolated cells in controlled laboratory conditions (also known as cell culture).


In vivo Latin for within the living. In biomedical research this refers to experiments that are done in a living organism. Experiments in model systems such as mice or fruit flies are an example of in vivo research.


Islets of Langerhans Clusters in the pancreas where insulin-producing beta cells live.


Macula A small spot at the back of the retina, densely packed with the rods and cones that receive light, which is responsible for high-resolution central vision.


Mesenchymal stem cells (MSCs) A term used to describe cells isolated from the connective tissue that surrounds other tissues and organs. MSCs were first isolated from the bone marrow and shown to be capable of making bone, cartilage and fat cells. MSCs are now grown from other tissues, such as fat and cord blood. Not all MSCs are the same and their characteristics depend on where in the body they come from and how they are isolated and grown. May also be called mesenchymal stromal cells.


Multipotent stem cells Stem cells that can give rise to several different types of specialized cells in specific tissues; for example, blood stem cells can produce the different types of cells that make up the blood, but not the cells of other organs such as the liver or the brain.


Neuron An electrically excitable cell that processes and transmits information through electrical and chemical signals in the central and peripheral nervous systems.


Pancreatic beta cells Cells responsible for making and releasing insulin, the hormone responsible for regulating blood sugar levels. Type I diabetes occurs when these cells are attacked and destroyed by the body's immune system.


Photoreceptors Rod or cone cells in the retina that receive light and send signals to the optic nerve, which passes along these signals to the brain.


Placebo A pill, injection or other treatment that has no therapeutic benefit; often used as a control in clinical trials to see whether new treatments work better than no treatment.


Placebo effect Perceived or actual improvement in symptoms that cannot be attributed to the placebo itself and therefore must be the result of the patient's (or other interested person's) belief in the treatment's effectiveness.


Pluripotent stem cells Stem cells that can become all the cell types that are found in an embryo, fetus or adult, such as embryonic stem cells or induced pluripotent (iPS) cells.


Preclinical research Laboratory research on cells, tissues and/or animals for the purpose of discovering new drugs or therapies.


Precursor cells An intermediate cell type between stem cells and differentiated cells. Precursor cells have the potential to give rise to a limited number or type of specialized cells. Also called progenitor cells.


Progenitor cells An intermediate cell type between stem cells and differentiated cells. Progenitor cells have the potential to give rise to a limited number or type of specialized cells and have a reduced capacity for self-renewal. Also called precursor cells.


Regenerative Medicine An interdisciplinary branch of medicine with the goal of replacing, regenerating or repairing damaged tissue to restore normal function. Regenerative treatments can include cellular therapy, gene therapy and tissue engineering approaches.


Reprogramming In the context of stem cell biology, this refers to the conversion of differentiated cells, such as fibroblasts, into embryonic-like iPS cells by artificially altering the expression of key genes.


Retinal pigment epithelium A single-cell layer behind the rods and cones in the retina that provide support functions for the rods and cones.


RNA Ribonucleic acid; it "reads" DNA and acts as a messenger for carrying out genetic instructions.


Scientific method A systematic process designed to understand a specific observation through the collection of measurable, empirical evidence; emphasis on measurable and repeatable experiments and results that test a specific hypothesis.


Self-renewal A special type of cell division in stem cells by which they make copies of themselves.


Somatic stem cells Scientific term for tissue-specific or adult stem cells.


Stem cells Cells that have both the capacity to self-renew (make more stem cells by cell division) and to differentiate into mature, specialized cells.


Stem cell tourism The travel to another state, region or country specifically for the purpose of undergoing a stem cell treatment available at that location. This phrase is also used to refer to the pursuit of untested and unregulated stem cell treatments.


TeratomaA benign tumor that usually consists of several types of tissue cells that are foreign to the tissue in which the tumor is located.


Tissue A group of cells with a similar function or embryological origin. Tissues organize further to become organs.


Tissue-specific stem cells Stem cells that can give rise to the specialized cells in specific tissues; blood stem cells, for example, can produce the different types of cells that make up the blood, but not the cells of other organs such as the liver or the brain. Includes all stem cells other than pluripotent stem cells such as embryonic and induced pluripotent cells. Also called adult or somatic stem cells.


Totipotent The ability to give rise to all the cells of the body and cells that arent part of the body but support embryonic development, such as the placenta and umbilical cord.


Translational research Research that focuses on how to use knowledge gleaned from basic research to develop new drugs, treatments or therapies.


Zygote The single cell formed when a sperm cell fuses with an egg cell.

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Stem Cell Glossary A Closer Look at Stem Cells

Stemming retinal regeneration with pluripotent stem cells …

JavaScript is disabled on your browser. Please enable JavaScript to use all the features on this page. Abstract

Cell replacement therapy is a promising treatment for irreversible retinal cell death in diverse diseases, such as age-related macular degeneration (AMD), Stargardt's disease, retinitis pigmentosa (RP) and glaucoma. These diseases are all characterized by the degeneration of one or two retinal cell types that cannot regenerate spontaneously in humans. Aberrant retinal pigment epithelial (RPE) cells can be observed through optical coherence tomography (OCT) in AMD patients. In RP patients, the morphological and functional abnormalities of RPE and photoreceptor layers are caused by a genetic abnormality. Stargardt's disease or juvenile macular degeneration, which is characterized by the loss of the RPE and photoreceptors in the macular area, causes central vision loss at an early age. Loss of retinal ganglion cells (RGCs) can be observed in patients with glaucoma. Once the retinal cell degeneration is triggered, no treatments can reverse it. Transplantation-based approaches have been proposed as a universal therapy to target patients with various concomitant diseases. Both the replacement of dead cells and neuroprotection are strategies used to rescue visual function in animal models of retinal degeneration. Diverse retinal cell types derived from pluripotent stem cells, including RPE cells, photoreceptors, RGCs and even retinal organoids with a layered structure, provide unlimited cell sources for transplantation. In addition, mesenchymal stem cells (MSCs) are multifunctional and protect degenerating retinal cells. The aim of this review is to summarize current findings from preclinical and clinical studies. We begin with a brief introduction to retinal degenerative diseases and cell death in diverse diseases, followed by methods for retinal cell generation. Preclinical and clinical studies are discussed, and future concerns about efficacy, safety and immunorejection are also addressed.

Retinal degeneration

Pluripotent stem cells

Retinal pigment epithelium

Stem cell-derived retinal cell

Retinal tissue


age-related macular degeneration

retinal pigment epithelial

retinal ganglion cells

mesenchymal stem cells

photoreceptor outer segment

pigment epithelium-derived factor

vascular endothelial growth factor

human embryonic stem cells

induced pluripotent stem cells

optical coherence tomography

visual evoked potential

adaptive optics scanning laser ophthalmoscopy

Toll-like receptor 3

genome-wide association analysis

retinal precursor cells

brain-derived neurotrophic factor

serum-free floating culture of embryoid body-like aggregates

hyaluronan and methylcellulose

regenerated wild Antheraeapernyi silk fibroin

human leukocyte antigen

outer limiting membrane

human Mller glia cells

inner segment/outer segment

adipose-derived stem cells

retinal pigment epithelial stem cells

central nervous system stem cells

membrane attack complexes

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2018 The Authors. Published by Elsevier Ltd.

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Stemming retinal regeneration with pluripotent stem cells ...

Current Strategies and Challenges for Purification of …

Theranostics 2017; 7(7):2067-2077. doi:10.7150/thno.19427


Kiwon Ban1, Seongho Bae2, Young-sup Yoon2, 3

1. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong; 2. Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia, USA; 3. Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea.

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license ( See for full terms and conditions.

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) are considered a most promising option for cell-based cardiac repair. Hence, various protocols have been developed for differentiating hPSCs into CMs. Despite remarkable improvement in the generation of hPSC-CMs, without purification, these protocols can only generate mixed cell populations including undifferentiated hPSCs or non-CMs, which may elicit adverse outcomes. Therefore, one of the major challenges for clinical use of hPSC-CMs is the development of efficient isolation techniques that allow enrichment of hPSC-CMs. In this review, we will discuss diverse strategies that have been developed to enrich hPSC-CMs. We will describe major characteristics of individual hPSC-CM purification methods including their scientific principles, advantages, limitations, and needed improvements. Development of a comprehensive system which can enrich hPSC-CMs will be ultimately useful for cell therapy for diseased hearts, human cardiac disease modeling, cardiac toxicity screening, and cardiac tissue engineering.

Keywords: Cardiomyocytes, hPSCs

Heart failure is the leading cause of death worldwide [1]. Approximately 6 million people suffer from heart failure in the United States every year [1]. Despite this high incidence, existing surgical and pharmacological interventions for treating heart failure are limited because these approaches only delay the progression of the disease; they cannot directly repair the damaged hearts [2]. In the case of large myocardial infarction (MI), patients progress to heart failure and die within short time from the onset of symptoms [3].

The adult human heart has minimal regenerative capacity, because during mammalian development, the proliferative capacity of cardiomyocytes (CMs) progressively diminishes and becomes terminally differentiated shortly after birth [4].Therefore, once CMs are damaged, they are rarely restored [5]. When MI occurs, the infarcted area is easily converted to non-contractile scar tissue due to loss of CMs and replacement by fibrosis [6]. Development of a fibroblastic scar initiates a series of events that lead to adverse remodeling, hypertrophy, and eventual heart failure [2, 3, 7].

While heart transplantation is considered the most viable option for treating advanced heart failure, the number of available donor hearts is always less than needed [6]. Therefore, more realistic therapeutic options have been required [2]. Accordingly, over the past two decades, cell-based cardiac repair has been intensively pursued [2, 7]. Several different cell types have been tested and varied outcomes were obtained. Indeed, the key factor for successful cell-based cardiac repair is to find the optimal cell type that can restore normal heart function. Naturally, CMs have been considered the best cell type to repair a damaged heart [8]. In fact, many scientists hypothesized that implanted CMs would survive in damaged hearts and form junctions with host CMs and synchronously contract with the host myocardium [9]. In fact, animal studies with primary fetal or neonatal CMs demonstrated that transplanted CMs could survive in infarcted hearts [9-11]. These primary CMs reduced scar size, increased wall thickness, and improved cardiac contractile function with signs of electro-mechanical integration [9-11]. These studies strongly suggest that CMs can be a promising source to repair the heart. However, the short supply and ethical concerns disallow using primary human CMs. In a patient with ischemic cardiomyopathy, about 40-50% of the CMs are lost in 40 to 60 grams of heart tissue [7]. Even if we seek to regenerate a fairly small portion of the damaged myocardium, a large number of human primary CMs would be required, which is impossible.

Accordingly, CMs differentiated from human pluripotent stem cells (hPSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have emerged as a promising option for candidate CMs for cell therapy [12, 13]. hPSCs have many advantages as a source for CMs. First, hPSCs have obvious cardiomyogenic potential. hPSC derived-CMs (hPSC-CMs) possess a clear cardiac phenotype, displaying spontaneous contraction, cardiac excitation-contraction (EC) coupling, and expression of cardiac transcription factors, cardiac ion channels, and cardiac structural proteins [14, 15]. Second, undifferentiated hPSCs and their differentiated cardiac progeny display significant proliferation capacity, allowing generation of a large number of hPSC-CMs. Lastly, many pre-clinical studies demonstrated that implantation of hPSC-CMs can repair injured hearts and improve cardiac function [16-19]. Histologically, implanted hPSC-CMs are engrafted, aligned and coupled with the host CMs in a synchronized manner [16-19].

In the last two decades, various protocols for differentiating hPSCs into CMs have been developed to improve the efficiency, purity and clinical compatibility [20] [18]. The reported differentiation methods include, but are not limited to: differentiation via embryoid body (EB) formation [20], co-culture with END-2 cells [18], and monolayer culture [15, 21, 22]. The EB-mediated CM differentiation protocol is one of the most widely employed methods due to its simple procedure and low cost. However, it often becomes labor-intensive to produce scalable EBs for further differentiation, which makes it difficult for therapeutic applications. EB-mediated differentiation also produces inconsistent results, showing beating CMs from 5% to 70% of EBs. Recently, researchers developed monolayer methods to complement the problems of EB-based methods [15, 21, 22]. In one representative protocol, hPSCs are cultured at a high density (up to 80%) and treated with a high concentration of Activin A (100 ng/ml) for 1 day and BMP4 (10 ng/ml) for 4 days followed by continuous culture on regular RPMI media with B27 [15]. This protocol induces spontaneous beating at approximately 12 days and produces approximately 40% CMs after 3 weeks. These hPSC-CMs can be further cultured in RPMI-B27 medium for another 2-3 weeks without significant cell damage [15]. However, these protocols use media with proprietary formulations, which complicates clinical application. As shown, most monolayer-based methods employ B27, which is a complex mix of 21 components. Some of the components of B27, including bovine serum albumin (BSA), are animal-derived products, and the effects of B27 components on differentiation, maturation or subtype specification processes are poorly defined. In 2014, Burridge and his colleagues developed an advanced protocol that is defined, cost-effective and efficient [22]. By subtracting one component from B27 at a time and proceeding with cardiac differentiation, the researchers reported that BSA and L-ascorbic acid 2-phosphate are essential components in cardiac differentiation. Subsequently, by replacing BSA with rice-derived recombinant human albumin, the chemically defined medium with 3 components (CDM3) was produced. The application of a GSK-inhibitor, CHIR99021, for the first 2 days followed by 2 days of the Wnt-inhibitor Wnt-59 to cells is an optimal culture condition in CDM3 resulting in similar levels of live-cell yields and CM differentiation [22].

Despite remarkable improvement in the generation of hPSC-CMs, obtaining pure populations of hPSC-CMs still remains challenging. Currently available methods can only generate a mixture of cells which include not only CMs but other cell types. This is one of the most critical barriers for applications of hPSC-CMs in regenerative therapy, drug discovery, and disease investigation. For Instance, cardiac transplantation of non-pure hPSC-CMs mixed with undifferentiated hPSCs or other cell types may produce tumors or unwanted cell types in hearts [23-28]. Accordingly, a pure or enriched population of hPSC-CMs would be required, particularly for cardiac cell therapy. Enriched hPSC-CMs would also be more beneficial for myocardial repair due to improved electric and mechanical properties [29]. A pure, homogeneous population of hPSC-CMs would pose less arrhythmic risk and have enhanced contractile performance, and would be more useful in disease modeling as they better reflect native CM physiology. Finally, purified hPSC-CMs would better serve for testing drug efficacy and toxicity. Therefore, many researchers have tried to develop methods to purify CMs from cardiomyogenically differentiated hPSCs.

There are three important topics that are not addressed in this review. First is the beneficial role of other cell types such as endothelial cells and fibroblasts in the integration, survival, and function of CMs [30-32]. We did not discuss this issue because it would need a separate review due to the volume of material. While the roles of such cells are important, the value of having purified hPSC-CMs is not diminished. Although cell mixtures or tissue engineered products can be used, unless purified CMs are employed, they would form tumors or other cells/tissues when implanted in vivo. Our point here is that even if cardiomyocytes are mixed with non-CMs, all cells should be clearly defined and purified as well. If the mixture is made in a non-purified or non-defined manner (for example, an unsophisticated top-down approach), there would be undefined cells that are neither CMs, ECs, nor fibroblasts and these unidentified cells will make aberrant tissues or tumors. Second, we did not deal with maturation of hPSC-CMs because of its broad scope and depth [33, 34]. Third is direct reprogramming or conversion of somatic cells into CMs. There has been another advancement in the generation of CMs by directly reprogramming or converting somatic cells into CM-like cells by introducing a combination of cardiac transcription factors (TFs) or muscle-specific microRNAs (miRNAs) both in vitro and in vivo [35-41]. These cells are referred to as induced CMs (iCMs) or cardiac-like myocytes (iCLMs). While this is an important advancement, we did not cover this topic either due to its size. Accordingly, this review will focus on the various strategies for purifying or enriching hPSC-CMs reported to date (Figure 1).

Early on, researchers isolated hPSC-CMs manually under microscopy by mechanically separating out the beating areas from myogenically differentiating hPSC cultures [18, 20, 42]. This method usually generates 5-70% hPSC-CMs. Although generally crude, it can enrich even higher percentages of CMs with further culture. This manual isolation method has the advantage of being easy, but while it can be useful for small-scale research, it is very labor intensive and not scalable, precluding large scale research or clinical application.

Currently available strategies for enriching cardiomyocytes derived from human pluripotent stem cells.

Xu et el. reported that hPSC-CMs, due to their physical and structural properties, can be enriched by Percoll density gradient centrifugation [43]. Percoll was first formulated by Pertoft et al [44] and it was originally developed for the isolation of cells, organelles, or viruses by density centrifugation. The Percoll-based method has several advantages. The procedure for Percoll-based separation is very simple and easy, it is inexpensive, and its low viscosity allows more rapid sedimentation and lower centrifugal forces compared to a sucrose density gradient. Lastly, it can be prepared and kept for a long time in an isotonic solution to maintain osmolarity. Although Percoll separation has resulted in major improvements in hPSC-CM isolation procedures, it has clear limitations with regard to purity and scalability. Previous studies found that Percoll separation is only able to enrich 40 -70% of hPSC-CMs. It is also not compatible with large-scale enrichment of hPSC-CMs.

Another traditional method for purifying hPSC-CMs is based on the expression of a drug resistant gene or a fluorescent reporter gene such as eGFP or DsRed, which is driven by a cardiac specific promoter in genetically modified hPSC lines [45, 46]. Here, enrichment of hPSC-CMs can be achieved by either drug treatment to eliminate cells that do not express the drug resistant gene or with FACS to isolate fluorescent cells [47, 48].

Briefly, enrichment of PSC-CMs by genetically based selection was first reported by Klug et al [49]. The authors generated murine ES cell lines via permanent gene transfection of the aminoglycoside phosphotransferase gene driven by the MHC (MYH7) promoter. With this approach, highly purified murine ESC-CMs up to 99% were achieved. Next, several studies reported the use of various CM-specific promoters to enrich ESC-CMs such as Mhc (Myh6), Myh7, Ncx (Sodium Calcium exchanger) and Mlc2v (Myl2) [46, 50, 51]. In the case of hESCs, MHC/EGFP hESCs were generated by permanent transfection of the EGFP-tagged MHC promoter [52]. Similarly, an NKX2.5/eGFP hESC line was generated to enrich GFP positive CMs [53]. However, since MHC and NKX2.5 are expressed in general CMs, the resulting CMs contain a mixture of the three subtypes of CMs, nodal-, atrial-, and ventricular-like CMs. To enrich only ventricular-like CMs, Huber et al. generated MLC2v/GFP ESCs to be able to isolate MLC2v/GFP positive ventricular-like cells by FACS [52] [54-57]. In addition, the cGATA6 gene was used to purify nodal-like hESC-CMs [58]. Future studies should focus on testing new types of cardiac specific promoters and devising advanced selection procedures to improve this strategy.

While fluorescence-based cell sorting is more widely used, the drug selection method may be a better approach to enrich high purity of hPSC-CMs during differentiation/culture as it does not require FACS. The advantage is its capability for high-purity cell enrichment due to specific gene-based cell sorting. These highly pure cells can allow more precise mechanistic studies and disease modeling. Despite its many advantages, the primary weakness of genetic selection is genetic manipulation, which disallows its use for therapeutic application. Insertion of reporter genes into the host genome requires viral or nonviral transfection/transduction methods, which can induce mutagenesis and tumor formation [50, 59-61].

Practically, antibody-based cell enrichment is the best method for cell purification to date. When cell type-specific surface proteins or marker proteins are known, one can tag cells with antibodies against the proteins and sort the target cells by FACS or magnetic-activated cell sorting (MACS). The main advantage is its specificity and sensitivity, and its utility is well demonstrated in research and even in clinical therapy with hematopoietic cells [62]. Another advantage is that multiple surface markers can be used at the same time to isolate target cells when one marker is not sufficient. However, no studies have reported surface markers that are specific for CMs, even after many years. Recently, though, several researchers demonstrated that certain proteins can be useful for isolating hPSC-CMs.

In earlier studies, KDR (FLK1 or VEGFR2) and PDGFR- were used to isolate cardiac progenitor cells [63]. However, since these markers are also expressed on hematopoietic cells, endothelial cells, and smooth muscle cells, they could not enrich only hPSC-CMs. Next, two independent studies reported two surface proteins, SIRPA [64] and VCAM-1 [65], which it was claimed could specifically identify hPSC-CMs. Dubois et al. screened a panel of 370 known antibodies against CMs differentiated from hESCs and identified SIRPA as a specific surface protein expressed on hPSC-CMs [64]. FACS with anti-SIRPA antibody enabled the purification of CMs and cardiac precursors from cardiomyogenically differentiating hPSC cultures, producing cardiac troponin T (TNNT2, also known as cTNT)-positive cells, which are generally considered hPSC-CMs, with up to 98% purity. In addition, a study performed by Elliot and colleagues identified another cell surface marker, VCAM1 [53]. In this study, the authors used NKX2.5/eGFP hESCs to generate hPSC-CMs, allowing the cells to be sorted by their NKX2.5 expression. NKX2.5 is a well-known cardiac transcription factor and a specific marker for cardiac progenitor cells [66, 67]. To identify CM-specific surface proteins, the authors performed expression profiling analyses and found that expression levels of both VCAM1 and SIRPA were significantly upregulated in NKX2.5/eGFP+ cells. Flow cytometry results showed that both proteins were expressed on the cell surface of NKX2.5/eGFP+ cells. Differentiation day 14 NKX2.5/eGFP+ cells expressed VCAM1 (71 %) or SIRPA (85%) or both VCAM1 and SIRPA (37%). When the FACS-sorted SIRPA-VCAM1-, SIRPA+ or SIRPA+VCAM1+ cells were further cultured, only SIRPA+ or SIRPA+VCAM1+ cells showed NKX2.5/eGFP+ contracting portion. Of note, NKX2.5/eGFP and SIRPA positive cells showed higher expression of smooth muscle cell and endothelial cell markers indicating that cells sorted solely based on SIRPA expression may not be of pure cardiac lineage. Hence, the authors concluded that a more purified population of hPSC-CMs could be isolated by sorting with both cell surface markers. Despite significant improvements, it appears that these surface markers are not exclusively specific for CMs as these antibodies also mark other cell types including smooth muscle cells and endothelial cells. Furthermore, they are also known to be expressed in the brain and the lung, which raises concerns whether these surface proteins can be used as sole markers for the purification of hPSC-CMs compatible for clinical applications.

More recently, Protze et al. reported successful differentiation and enrichment of sinoatrial node-like pacemaker cells (SANLPCs) from differentiating hPSCs by using cell surface markers and an NKX2-5-reporter hPSC line [68]. They found that BMP signaling specified cardiac mesoderm toward the SANLPC fate and retinoic acid signaling enhanced the pacemaker phenotype. Furthermore, they showed that later inhibition of the FGF pathway, the TFG pathway, and the WNT pathway shifted cell fate into SANLPCs, and final cell sorting for SIRPA-positive and CD90-negative cells resulted in enrichment of SANLPCs up to ~83%. These SIRPA+CD90- cells showed the molecular, cellular and electrophysiological characteristics of SANLPCs [68]. While this study makes important progress in enriching SANLPCs by modulating signaling pathways, no specific surface markers for SANLPCs were identified and the yield was still short of what is usually expected for cells purified via FACS.

Hattori et al. developed a highly efficient non-genetic method for purifying hPSC-derived CMs, in which they employed a red fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), that can label active mitochondria. Since CMs contain a large number of mitochondria, CMs from mice and marmosets (monkey) could be strongly stained with TMRM [69]. They further found that primary CMs from several different types of animals and CMs derived from both mESCs and hESCs were successfully purified by FACS up to 99% based on the TMRM signals. In addition to its efficiency for CM enrichment, TMRM did not affect cell viability and disappeared completely from the cells within 24 hrs. Importantly, injected hPSC-CMs purified in this way did not form teratoma in the heart tissues. However, since TMRM only functions in CMs with high mitochondrial density, this method cannot purify entire populations of hPSC-CMs [64]. While originally TMRM was claimed to be able to isolate mature hPSC-CMs, mounting evidence indicates that hPSC-CMs are similar to immature human CMs at embryonic or fetal stages. Therefore, both the exact phenotype of the cells isolated by TMRM and its utility are rather questionable [33, 34]. Two subsequent studies demonstrated that TMRM failed to accurately distinguish hPSC-CMs due to the insufficient amounts of mitochondria [64].

Employing the unique metabolic properties of CMs, Tohyama et al. developed an elegant purification method to enrich PSC-CMs [70]. This approach is based on the remarkable biochemical differences in lactate and glucose metabolism between CMs and non-CMs, including undifferentiated cells. Mammalian cells use glucose as their main energy source [71]. However, CMs are capable of energy production from different sources such as lactate or fatty acids [71]. A comparative transcriptome analysis was performed to detect metabolism-related genes which have different expression patterns between newborn mouse CMs and undifferentiated mouse ESCs. These results showed that CMs expressed genes encoding tricarboxylic acid (TCA) cycle enzymes more than genes related to lipid and amino acid synthesis and the pentose phosphate cycle compared to undifferentiated ESCs. To further prove this observation, they compared the metabolites of these pathways using fluxome analysis between CMs and other cell types such as ESCs, hepatocytes and skeletal muscle cells, and found that CMs have lower levels of metabolites related to lipid and amino acid synthesis and pentose phosphate. Subsequently, authors cultured newborn rat CMs and mouse ESCs in media with lactate, forcing the cells to use the TCA cycle instead of glucose, and they observed that CMs were the only cells to survive this condition for even 96 hrs. They further found that when PSC derivatives were cultured in lactate-supplemented and glucose-depleted culture medium, only CMs survived. Their yield of CM population was up to 99% and no tumors were formed when these CMs were transplanted into hearts. This lactate-based method has many advantages: its simple procedures, ease of application, no use of FACS for cell sorting, and relatively low cost. More recently, this method was applied to large-scale CM aggregates to ensure scalability. As a follow-up study, the same group recently reported a more refined lactate-based enrichment method which further depletes glutamine in addition to glucose [72]. The authors found that glutamine is essential for the survival of hPSCs since hPSCs are highly dependent on glycolysis for energy production rather than oxidative phosphorylation. The use of glutamine- and glucose-depleted lactate-containing media resulted in more highly purified hPSC-CMs with less than 0.001% of residual PSCs [72]. One concern of this lactate-based enrichment method is the health of the purified hPSC-CMs, because physiological and functional characteristics of hPSC-CMs cultured in glucose- and glutamine-depleted media for a long time may have functional impairment since CMs with mature mitochondria were not able to survive without glucose and glutamine, although they were able to use lactate to synthesize pyruvate and glutamate [72]. In addition, this lactate-based strategy can only be applied to hPSC- CMs, but not other hPSC derived cells such as neuron or -cells.

Our group also recently reported a new method to isolate hPSC-CMs by directly labelling cardiac specific mRNAs using nano-sized probes called molecular beacons (MBs) [29, 73, 74]. Designed to detect intracellular mRNA targets, MBs are dual-labeled antisense oligonucleotide (ODN) nano-scale probes with a DNA or RNA backbone, a Cy3 fluorophore at the 5' end, and a Black Hole quencher 2 (BHQ2) at the 3' end [75, 76]. They form a stem-loop (hairpin) structure in the absence of a complementary target, quenching the fluorescence of the reporter. Hybridization with the target mRNA opens the hairpin and physically separates the reporter from the quencher, allowing a fluorescence signal to be emitted upon excitation. The MB-based method can be applied to the purification of any cell type that has known specific gene(s) [77].

In one study [29], we designed five MBs targeting unique sites in TNNT2 or MYH6/7 mRNA in both mouse and human. To determine the most efficient transfection method to deliver MBs into living cells, various methods were tested and nucleofection was found to have the highest efficiency. Next, we tested the sensitivity and specificity of MBs using an immortalized mouse CM cell line, HL-1, and other cell types. Finally, we narrowed it down to one MB, MHC-MB, which showed >98% sensitivity and > 95% specificity. This MHC-MB was applied to cardiomyogenically differentiated mouse and human PSCs and FACS sorting was performed. The resultant MHC-MB-positive cells expressed cardiac proteins at ~97% when measured by flow cytometry. These sorted cells also demonstrated spontaneous contraction and all the molecular and electrophysiological signatures of human CMs. Importantly, when these purified CMs were injected into the mouse infarcted myocardium, they were well integrated into the myocardium without forming any tumors, and they improved cardiac function.

In a subsequent study [74], we refined a method to enrich ventricular CMs from differentiating PSCs (vCMs) by targeting a transcription factor which is not robustly expressed in cells. Since vCMs are the main source for generating cardiac contractile forces and the most frequently damaged in the heart, there has been great demand to develop a method that can obtain a pure population of vCMs for cardiac repair. Despite this critical unmet need, no studies have demonstrated the feasibility of isolating ventricular CMs without permanently altering their genome. Accordingly, we first designed MBs targeting the Iroquois homeobox protein 4 (Irx4) mRNA, a vCM specific transcription factor [78, 79]. After testing sensitivity and specificity, one IRX4-MB was selected and applied to myogenically differentiated mPSCs. The FACS-sorted IRX4-MB-positive cells exhibited vCM-like action potentials in more than 98% of cells when measured by several electrophysiological analyses including patch clamp and Ca2+ transient analyses. Furthermore, these cells maintained spontaneous contraction and expression of vCM-specific proteins.

The MB-based cell purification method is theoretically the most broadly applicable technology among the purification methods because it can isolate any target cells expressing any specific gene. Thus, the MB-based sorting technique can be applied to the isolation of other cell types such as neural-lineage cells or islet cells, which are critical elements in regenerative medicine but do not have specific surface proteins identified to date. In addition, theoretically, this technology may have the highest efficiency when MBs are designed to have the maximum sensitivity and specificity for the cells of interest, but not others. These characteristics are particularly important for cell therapy. Despite these advantages, the delivery method of MB into the cells needs to be improved. So far, nucleofection is the best delivery method, but caused some cell damage with < 70% cell viability. Thus, development of a safer delivery method will enable wider application of MB-based cell enrichment.

Recently, Miki and colleagues reported a novel method for purifying cells of interest based on endogenous miRNA activity [80]. Miki et al. employed several synthetic mRNA switches (= miRNA switch), which consist of synthetic mRNA sequences that include a recognition sequence for miRNA and an open reading frame that codes a desired gene, such as a regulatory protein that emits fluorescence or promotes cell death. If the miRNA recognition sequence binds to miRNA expressed in the desired cells, the expression of the regulatory protein is suppressed, thus distinguishing the cell type from others that do not contain the miRNA and express the protein.

Briefly, the authors first identified 109 miRNA candidates differentially expressed in distinct stages of hPSC-CMs (differentiation day 8 and 20). Next, they found that 14 miRNAs were co-expressed in hPSC-CMs at day 8 and day 20 and generated synthetic mRNAs that recognize these 14 miRNA, called miRNA switches. Among those miRNA switches, miR-1-, miR-208a-, and miR-499a-5p-switches successfully enriched hPSC-CMs with purity of sorted cells up to 96% determined by TNNT2 intracellular flow cytometry. Particularly, hPSC-CMs enriched by the miR-1-switch showed substantially higher expression of several cardiac specific genes/proteins and lower expression of non-CM genes/proteins compared with control cells. Patch clamp confirmed that these purified hPSC-CMs possessed both ventricular-like and atrial-like action potentials.

One of the major advantages of this technology is its wider applicability to other cell types. miRNA switches have the flexibility to design the open reading frame in the mRNA sequence such that any desired transgene can be incorporated into the miRNA switches to regulate the cell phenotype based on miRNA activity. The authors tested this possibility by incorporating BIM sequence, an apoptosis inducer, into the cardiac specific miR-1- and miR-208a switches and tested whether they could selectively induce apoptosis in non-CMs. They found that miR-1- and miR-208a-Bim-switches successfully enriched cTNT-positive hPSC-CMs without cell sorting. Enriched hPSC-CMs by 208a-Bim-switch were injected into the hearts of mice with acute MI and they engrafted, survived, expressed both cTNT and CX43, and formed gap junctions with the host myocardium. No teratoma was detected. In addition, other miRNA switches such as miR-126-, miR-122-5p-, and miR-375-switches targeting endothelial cells, hepatocytes, and -cells, respectively, successfully enriched these cell types differentiated from hPSCs. However, identification of specific miRNAs expressed only in the specific cell type of interest and verification of their specificity in target cells will be key issues for continuing to use this miRNA-based cell enrichment method.

Recent advances in biomedical engineering have contributed to developing systems that can isolate target cells using physicochemical properties of the cells. Microfluidic systems have been intensively applied for cell separation due to recent improvements in miniaturizing a cell culture system [81-83]. These advances made possible the design of automated microfluidic devices with cellular microenvironments and controlled fluid flows that save time and cost in experiments. Thus, there have been an increasing number of studies seeking to apply the microfluidic system for cell separation. Among the first, Singh et al. tested the possibility of using a microfluidic system for the separation of hPSC [84] by preparative detachment of hPSCs from differentiating cultures based on differences in the adhesion properties of different cell types. Distinct streams of buffer that generated varying levels of shear stress further allowed selective enrichment of hPSC colonies from mixed populations of adherent non-hPSCs, achieving up to 95% purity. Of note, this strategy produced hPSC survival rates almost two times higher than FACS, reaching 80%.

Subsequently, for hPSC-CMs purification, Xin et al. developed a microfluidic system with integrated ridge-like flow derivations and fishnet-like microcolumns for the enrichment of hiPSC-CMs [85]. This device is composed of a 250 mm-long microfluidic channel, which has two integrated parallel microcolumns with surfaces functionalized with anti-human TRA-1 antibody for undifferentiated hiPSC trapping. Aided by the ridge-like surface patterns on the upper wall of the channel, micro-streams are generated so that the cell suspension of mixed undifferentiated hiPSCs and hiPSC-CMs are forced to cross the functionalized fishnet-like microcolumns, resulting in trapping of undifferentiated hiPSCs due to the interaction between the hiPSCs and the columns, and the untrapped hiPSC-CMs are eventually separated. By modulating flow and coating with anti-human TRA-1 antibody, they were able to enrich CMs to more than 80% purity with 70% viability. While this study demonstrated that a microfluidic device could be used for purifying hPSC-CMs, it was not realistic because the authors used a mixture of only undifferentiated hiPSCs and hiPSC-CMs. In real cardiomyogenically differentiated hiPSCs, undifferentiated hiPSCs are rare and many intermediate stage cells or other cell types are present, so the idea that this simple device can select only hiPSC-CMs from a complex mixture is uncertain.

Overall, the advantages of microfluidic system based cell isolation include fast speed, improved cell viability and low cost owing to the automated microfluidic devices that can control cellular microenvironments and fluid flows [86-88]. However, microfluidic-based cell purification methods have limitations in terms of low purity and scalability [89-92]. In fact, there have been only a few studies demonstrating the feasibility that microfluidic device-based cell separation could achieve higher than 80% purity of target cells. Furthermore, currently available microfluidic devices allow only separation of a small number of cells (< 1011). To employ microfluidic devices for large-scale cell production, we need to develop a next generation of microfluidic devices that can achieve a throughput greater than 1011 sorted cells per hour with > 95% purity.

Having available a large quantity of a homogeneous population of cells of interest is an important factor in advancing biomedical research and clinical medicine, and is especially true for hPSC-CMs. While remarkable progress has been made in the methods for differentiating hPSCs into CMs, technologies to enrich hPSC-CMs, particularly those which are clinically applicable, have been emerging only over the last few years. Contamination with other cell types and even the heterogeneous nature of hPSC-CMs significantly hinder their use for several future applications such as cardiac drug toxicology screening, human cardiac disease modeling, and cell-based cardiac repair. For instance, cardiac drug-screening assays require pure populations of hPSC-CMs, so that the observed signals can be attributed to effects on human CMs. Studies of human cardiac diseases can also be more adequately interpreted with purified populations of patient derived hiPSC-CMs. Clinical applications with hPSC-CMs will need to be free of other PSC derivatives to minimize the risk of teratoma formation and other adverse outcomes.

Summary of representative methods for hPSC-CM purification

Schematic pictures of microfluidic device for enriching hiPSC-CMs. (A) The part of the device designed for trapping undifferentiated hiPSCs. (B) (Left) Illustration of the overall microfluidic device assembled with peristaltic pump, cell suspension reservoirs, and a serpentine channel. (Right) Magnified image showing a channel combining microcolumns and ridge-like flow derivation structures. Modified from Li et al. On chip purification of hiPSC-derived cardiomyocytes using a fishnet-like microstructure. Biofabrication. 2016 Sep 8;8(3): 035017

Therefore, development of reproducible, effective, non-mutagenic, scalable, and economical technologies for purifying hPSC-CMs, independent of hPSC lines or differentiation protocols, is a fundamental requirement for the success of hPSC-CM applications. Fortunately, new technologies based on the biological specificity of CMs such as MITO-tracker, molecular beacons, lactate-enriched-glucose depleted-media, and microRNA switches have been developed. In addition, technologies based on engineering principles have recently yielded a promising platform using microfluidic technology. While due to the short history of this field, more studies are needed to verify the utility of these technologies, the growing attention toward this research is a welcome move.

Another important question raised recently is how to non-genetically purify chamber-specific subtypes of CMs such as ventricular-like, atrial-like and nodal-like hPSC-CMs. So far, only a few studies have addressed this potential with human PSCs. We also showed that a molecular beacon-based strategy could enrich ventricular CMs differentiated from PSCs [74]. Another study demonstrated generation of SA-node like pacemaker cells by using a stepwise treatment of various morphogens and small molecules followed by cell sorting with several sub-specific surface markers. However, the yield of both studies was relatively low (<85%). Given the growing clinical importance of chamber-specific CMs, the strategies for purifying specific subtypes of CM that are independent of hPSC lines or differentiation protocols should be continuously developed. A recently reported cell surface capture-technology [93, 94] may facilitate identification of chamber specific CM proteins that will be useful for target CM isolation.

In summary, technological advances in the purification of hPSC-CMs have opened an avenue for realistic application of hPSC-CMs. Although initial success was achieved for purification of CMs from differentiating hPSC cultures, questions such as scalability, clinical compatibility, and cellular damage remain to be answered and isolation of human subtype CMs has yet to be demonstrated. While there are other challenges such as maturity, in vivo integration, and arrhythmogenecity, this development of purification technology represents major progress in the field and will provide unprecedented opportunities for cell-based therapy, disease modeling, drug discovery, and precision medicine. Furthermore, the availability of chamber-specific CMs with single cell analyses will facilitate more sophisticated investigation of human cardiac development and cardiac pathophysiology.

This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP) (No 2015M3A9C6031514), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI15C2782, HI16C2211) and grants from NHLBI (R01HL127759, R01HL129511), NIDDK (DP3-DK108245). This work was also supported by a CityU Start-up Grant (No 7200492), a CityU Research Project (No 9610355), and a Georgia Immuno Engineering Consortium through funding from Georgia Institute of Technology, Emory University, and the Georgia Research Alliance.

The authors have declared that no competing interest exists.

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Current Strategies and Challenges for Purification of ...

IPSCjun19 Induced Pluripotent Stem Cells: differentiation …



This a hands-on practical course on Induced pluripotent stem cells (iPSCs) differentiation into hepatocytes.iPSCs provide an inexhaustible source of cells, which can bedifferentiated into any lineage. iPSCs are generated from normal and disease adult cells (such asblood cells or skin cells) via reprogramming using a defined set of transcription factors (Oct4, Sox2, c-Myc, Kif4).Hepatocytes are liver cells, which make up 70-85 % of the liver mass. These cells are involved inprocesses such as protein synthesis, carbohydrate metabolism and detoxification, which areassociated with many diseases. iPSC derived hepatocytes can be used for basic liver research such asunderstanding liver development and cell biology, as well as for disease modelling and toxicity screening.

In this Induced pluripotent stem cells (iPSCs) differentiation into hepatocytes courses:

1. Participants will be introduced to the background, maintenance and applications of iPSC technology. 2. During the hands-on practical, participants will learn how to derive hepatocytes from human iPSCs. 3. Participants will take part in interactive tutorials, Q&A clinics, and panel discussions, with leading scientistswho will answer any questions you may have about your individual projects, to help you to avoid the commonpitfalls of using iPSCs in your experiments.






1. To understand the history, manipulation and use of iPSCs 2. To gain knowledge of hepatocyte structure and function 3. Learn how to differentiate human iPSCs into hepatocytes 4. To gain knowledge about the use of differentiated hepatocytes in both basic research anddisease modelling






Professor David Hay

MRC Centre for Regenerative Medicine, University of Edinburgh


David Hay is Professor of Tissue Engineering at the University of Edinburgh. David has worked in the field of stem cell biology and differentiation for over fifteen years. David andhis team have highlighted the important role that pluripotent stem cells have to play inmodelling human liver biology in a dish and supporting failing liver function in vivo. Theimpact of this work has led to a number of peer reviewed publications, regular appearancesat high profile conferences and three start-up companies.

Dr Rute Tomaz

Wellcome-MRC Cambridge,Stem Cell Institute and the Department of Surgery


Dr Rute Tomaz is a postdoc research associate at Dr Ludovic Valliers lab, part of theWellcome-MRC Cambridge Stem Cell Institute and the Department of Surgery. Since joiningthe lab in 2016, her research has focused on the developing novel methods fordifferentiation and maturation of hepatocytes from human pluripotent stem cells forsubsequent applications such as disease modelling and drug screening. Prior to joining thislab, Rute did her PhD at Imperial College London where she studied gene regulationmechanisms in early cell fate choices using mouse embryonic stem cells as a model.

Dr Daniel Ortmann

Wellcome-MRC Cambridge, Stem Cell Institute and the Department of Surgery


Dr Florian Merkle

Principal Investigator, Metabolic Research Laboratories, University of Cambridge


The Merkle laboratory studies the molecular and cellular basis of human diseases using a combination of human cellular models and animal models. They have a particular interest in obesity, which leads to millions of premature deaths each year, lacks broadly effective treatments, and is associated with the aberrant function of specific neuron types in the hypothalamus. The lab developed methods to differentiate human pluripotent stem cells (hPSCs) into functional hypothalamic cell types in culture, enabling us to study their function in health and obesity using a range of cutting-edge techniques including genome engineering, single-cell transcriptomics, quantitative peptidomics, high content imaging, calcium imaging, and xenotransplantation. Research in the Merkle laboratory revolves around three areas: 1) Basic biology of human hypothalamic neurons 2) Genetic and environmental contributions to obesity 3) Translation and in vivo models Dr. Merkle is affiliated to the Cambridge Stem Cell Institute,collaborates with the Wellcome Sanger Institute and EBI in the context of his single-cell RNAseq work, and was recently awarded the prestigious Sir Henry Dale fellowship, during which he will use gene editing to explore how obesity-associated mutations alter cellular phenotypes.

Dr Annika Asplund

Senior Scientist, Takara Bio


Annika Asplund is a Senior Research Scientist at Takara Bio Europe, AB in Sweden. She completed her PhD in molecular medicine within the field of cardiovascular prevention at Gothenburg University, Sweden. In 2012 she joined Takara Bio, focusing on hepatocyte differentiation and maturation from human pluripotent stem cells. Annika has extensive experience in protocol development for hepatocyte differentiation and characterization as well as dissociation and cryopreservation and contributed to the development of several innovative solutions offered by Takara Bio. She is committed to improving Takara Bios hepatocyte solutions and to helping customers succeeding with their hepatocyte related experiments.

Dr Johannes Elvin

Senior Production and Service Specialist, Takara Bio


Johannes Elvin is a Senior Production and Service Specialist and joined Takara Bio Europe AB in 2018. He did his PhD at Gothenburg University in renal medicine and transitioned into the field of diabetes and obesity during his postdoc. He has been working extensively with in vitro cell culture, lentiviral transduction and protein expression modification. He is now handling and running service projects aimed at reprogramming somatic cells into pluripotent stem cells.

Dr Christian Andersson

Product Development Manager, Takara Bio


Christian Andersson is a senior scientist and Product Development Manager at Takara Bio Europe, AB. He has extensive experience of culturing and differentiate stem cells into preferred cell types, where his field of expertise are Beta Cells. He has worked in project team to commercialize, market and promote Takara Bios stem cell portfolio all over the world. He is part of the companys stem cell strategy and marketing team, defining stem cell projects, marketing approaches, and business development opportunities.








With this course you get our exclusive CamBioScience membership for free and receive an automatic 10% discount on your registration fee.

* These fees include your 10% discount.


To register, please click below and fill in the registration form. You will then receive an email confirmation in which you will be able to choose a payment method (pay by card online, or receive an invoice to process payment by bank transfer).






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IPSCjun19 Induced Pluripotent Stem Cells: differentiation ...

Induced pluripotent stem cells don’t increase genetic …

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 don't increase genetic ...

Induced Pluripotent Stem Cells – Embryo Project

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

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)

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Induced Pluripotent Stem Cells - Embryo Project