hESC-derived Cells Research Articles

Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering

Human embryonic stem cells (hESC) are promising for use in regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there have been no reports on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESC were cultured to form embryoid bodies (EB), and the MSC were then migrated out of the EB. hESCd-MSC had surface markers characteristic of MSC, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSC were encapsulated in alginate at a density of 1millioncellsml−1, with an average microbead size of 207μm. CPC contained mannitol porogen to create a porosity of 64% and 218-μm macropores, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSC encapsulated in microbeads in CPC had good viability from 1 to 21days. ALP gene expression at 21days was 25-fold that at 1day. Osteocalcin (OC) at 21days was two orders of magnitude of that at 1day. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21days was fivefold that at 1day. Mineral synthesis by the encapsulated hESCd-MSC at 21days was sevenfold that at 1day. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSC were encapsulated in alginate microbeads in macroporous CPC, showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications.

Generation and clonal isolation of retinal stem cells from human embryonic stem cells

Retinal stem cells (RSCs) are present within the pigmented ciliary epithelium (CE) of the adult human eye and produce progeny that differentiate in vitro into all neural retinal subtypes and retinal pigmented epithelium (RPE). We hypothesized that a RSC population, similar to the adult CE-derived RSC, is contained within pigmented colonies that arise in long-term cultures of human embryonic stem cells (hESCs) suggested to recapitulate retinal development in vitro. Single pigmented hESC-derived cells were isolated and plated in serum-free media containing growth factors and, after 2 weeks, clonal sphere colonies containing both pigmented and non-pigmented cells were observed. These colonies expressed the early retinal transcription factors Rx, Chx10 and Pax6, and could be dissociated and replated as single cells to form secondary clonal colonies. When allowed to differentiate, expression of markers for both RPE and neurons was observed. Rhodopsin expression was detected after explant co-culture and transplantation into the developing mouse eye as well as following treatment with soluble factors in vitro. We show that RSCs emerge in an in vitro model of retinal development and are a potential source of human photoreceptors for use in transplantation.

Prolonged Maturation Culture Favors a Reduction in the Tumorigenicity and the Dopaminergic Function of Human ESC‐Derived Neural Cells in a Primate Model of Parkinson's Disease

For the safe clinical application of embryonic stem cells (ESCs) for neurological diseases, it is critical to evaluate the tumorigenicity and function of human ESC (hESC)-derived neural cells in primates. We have herein, for the first time, compared the growth and function of hESC-derived cells with different stages of neural differentiation implanted in the brains of primate models of Parkinson's disease. We herein show that residual undifferentiated cells expressing ESC markers present in the cell preparation can induce tumor formation in the monkey brain. In contrast, a cell preparation matured by 42-day culture with brain-derived neurotrophic factor/glial cell line-derived neurotrophic factor (BDNF/GDNF) treatment did not form tumors and survived as primarily dopaminergic (DA) neurons. In addition, the monkeys with such grafts showed behavioral improvement for at least 12 months. These results support the idea that hESCs, if appropriately matured, can serve as a source for DA neurons without forming any tumors in a primate brain.

Spontaneous origin from human embryonic stem cells of liver cells displaying conjoint meso-endodermal phenotype with hepatic functions

Understanding the identity of lineage-specific cells arising during manipulations of stem cells is necessary for developing their potential applications. For instance, replacement of crucial functions in organ failure by transplantation of suitable stem-cell-derived cells will be applicable to numerous disorders, but requires insights into the origin, function and fate of specific cell populations. We studied mechanisms by which the identity of differentiated cells arising from stem cells could be verified in the context of natural liver-specific stem cells and whether such differentiated cells could be effective for supporting the liver following cell therapy in a mouse model of drug-induced acute liver failure. By comparing the identity of naturally occurring fetal human liver stem cells, we found that cells arising in cultures of human embryonic stem cells (hESCs) recapitulated an early fetal stage of liver cells, which was characterized by conjoint meso-endoderm properties. Despite this fetal stage, hESC-derived cells could provide liver support with appropriate metabolic and ammonia-fixation functions, as well as cytoprotection, such that mice were rescued from acute liver failure. Therefore, spontaneous or induced differentiation of human embryonic stem cells along the hepatic endoderm will require transition through fetal-like stages. This offers opportunities to prospectively identify whether suitable cells have been generated through manipulation of stem cells for cell therapy and other applications.

Embryonic stem cell trials for macular degeneration: a preliminary report

It has been 13 years since the discovery of human embryonic stem cells (hESCs). Our report provides the first description of hESC-derived cells transplanted into human patients. We started two prospective clinical studies to establish the safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium (RPE) in patients with Stargardt's macular dystrophy and dry age-related macular degeneration--the leading cause of blindness in the developed world. Preoperative and postoperative ophthalmic examinations included visual acuity, fluorescein angiography, optical coherence tomography, and visual field testing. These studies are registered with ClinicalTrials.gov, numbers NCT01345006 and NCT01344993. Controlled hESC differentiation resulted in greater than 99% pure RPE. The cells displayed typical RPE behaviour and integrated into the host RPE layer forming mature quiescent monolayers after transplantation in animals. The stage of differentiation substantially affected attachment and survival of the cells in vitro after clinical formulation. Lightly pigmented cells attached and spread in a substantially greater proportion (>90%) than more darkly pigmented cells after culture. After surgery, structural evidence confirmed cells had attached and continued to persist during our study. We did not identify signs of hyperproliferation, abnormal growth, or immune mediated transplant rejection in either patient during the first 4 months. Although there is little agreement between investigators on visual endpoints in patients with low vision, it is encouraging that during the observation period neither patient lost vision. Best corrected visual acuity improved from hand motions to 20/800 (and improved from 0 to 5 letters on the Early Treatment Diabetic Retinopathy Study [ETDRS] visual acuity chart) in the study eye of the patient with Stargardt's macular dystrophy, and vision also seemed to improve in the patient with dry age-related macular degeneration (from 21 ETDRS letters to 28). The hESC-derived RPE cells showed no signs of hyperproliferation, tumorigenicity, ectopic tissue formation, or apparent rejection after 4 months. The future therapeutic goal will be to treat patients earlier in the disease processes, potentially increasing the likelihood of photoreceptor and central visual rescue. Advanced Cell Technology.

A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification

The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in infants. Although it is well established that MLL-AF4 arises prenatally during human development, its effects on hematopoietic development in utero remain unexplored. We have created a human-specific cellular system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs). Functional studies, clonal analysis and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic, functional and gene expression impact. MLL-AF4 acts as a global transcriptional activator and a positive regulator of homeobox gene expression in hESCs. Functionally, MLL-AF4 enhances the specification of hemogenic precursors from hESCs but strongly impairs further hematopoietic commitment in favor of an endothelial cell fate. MLL-AF4 hESCs are transcriptionally primed to differentiate towards hemogenic precursors prone to endothelial maturation, as reflected by the marked upregulation of master genes associated to vascular-endothelial functions and early hematopoiesis. Furthermore, we report that MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells. This work illustrates how hESCs may provide unique insights into human development and further our understanding of how leukemic fusion genes, known to arise prenatally, regulate human embryonic hematopoietic specification.

Evaluation of Human Embryonic Stem Cell-derived Hepatocyte-like Cells for Detection of CYP1A Inducers

There is a great deal of interest in differentiation of human embryonic stem cells (hESCs) into hepatocyte-like cells for application in pharmaceutical screening. Cytochrome P450 (CYP) 1A is involved in the metabolic activation of procarcinogenic compounds as well as in detoxification of drugs. We differentiated hESCs into hepatocyte-like cells (hESC-derived hepatocyte-like cells) and examined whether CYP1A was induced in these cells by typical inducers of CYP1A. hESC-derived hepatocyte-like cells expressed albumin, α-fetoprotein, CYP3A4, CYP3A7, CYP1A1, CYP1A2, and UDP-glucuronyl transferase (UGT) 1A1 mRNA. The levels of CYP1A1, CYP1A2, and UGT1A1 mRNA expression were increased by omeprazole and 3-methylcholanthrene. Furthermore, the enzyme activity of CYP1A was also increased by these compounds. In conclusion, hESC-derived hepatocyte-like cells are available for the detection of CYP1A inducers.

The Potential of Human Embryonic Stem Cells for Articular Cartilage Repair and Osteoarthritis Treatment

Osteoarthritis is a debilitating joint disease present in epidemic proportions worldwide. Osteoarthritis results from degeneration of the articular cartilage of the joint surfaces due to acute trauma, or chronic wear and tear. Due to limited ability of cartilage to repair itself, and lack of available treatments, there is an urgent need for development of approaches to repair articular cartilage damage due to injury or osteoarthritic disease. Cell-based repair strategies are among the most promising of these approaches. Various adult cell sources for cartilage repair are proposed including autologous adult chondrocytes as well as adult Mesenchymal Stem Cells (MSC). Disadvantages such as destructive harvest protocols; poor proliferation, and particularly for MSC, considerable cellular heterogeneity, have limited success of these cell types for cartilage repair. Chondrogenic cells derived from human embryonic stem cells(hESC) offer a highly proliferative cell source, which when directed into the chondrogenic lineage, could provide an ideal source of cells for cartilage repair. Chondrogenic cells derived from human induced pluripotent stem cells(iPSC) offer additional advantages for patient-specific therapy. Recently protocols have been established for directed differentiation of hESC into the chondrogenic lineage. Harnessing the potential of hESC-derived chondrogenic cells will require comprehensive testing of their efficacy for in vivo cartilage repair, as well as considerations of safety and immunogenicity of the cells. Use of pro-chondrogenic factors and/or bioactive scaffolds may assist in optimizing cartilage repair by chondrogenic cells. Repair of cartilage damage in osteoarthritis is a special challenge because of the widespread damage and presence of signals and stressors which disrupt normal joint homeostasis. Particular promise in cell-based repair of osteoarthritis may be provided by chondrogenic progenitor cells which may mimic endogenous repair responses. This review discusses the current status of cell-based cartilage repair strategies and in particular the potential role of hESC-derived chondrocytes or chondroprogenitor cells for treatment of articular cartilage damage due to injury and osteoarthritis.

Challenges to the clinical application of pluripotent stem cells: towards genomic and functional stability

Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and are pluripotent, retaining the ability to differentiate into all cell types in the body. As a renewable source of various types of human cells, hESCs hold great therapeutic potential. Although significant advances have been achieved in defining the conditions needed to differentiate hESCs into various types of biologically active cells, many challenges remain in the clinical development of hESC-based cell therapy, such as the immune rejection of allogeneic hESC-derived cells by recipients. Breakthroughs in the generation of induced pluripotent stem cells (iPSCs), which are reprogrammed from somatic cells with defined factors, raise the hope that autologous cells derived from patient-specific iPSCs can be transplanted without immune rejection. However, recent genomic studies have revealed epigenetic and genetic abnormalities associated with induced pluripotency, a risk of teratomas, and immunogenicity of some iPSC derivatives. These findings have raised safety concerns for iPSC-based therapy. Here, we review recent advances in understanding the genomic and functional stability of human pluripotent stem cells, current challenges to their clinical application and the progress that has been made to overcome these challenges.

Theranostic effect of serial manganese‐enhanced magnetic resonance imaging of human embryonic stem cell derived teratoma

Although human embryonic stem cell (hESC) hold therapeutic potential, teratoma formation has deterred clinical translation. Manganese (Mn(2+)) enters metabolically active cells through voltage-gated calcium channels and subsequently, induces T(1) shortening. We hypothesized that serial manganese-enhanced MRI would have theranostic effect to assess hESC survival, teratoma formation, and hESC-derived teratoma reduction through intracellular accumulation of Mn(2+). Firefly luciferase transduced hESCs (hESC-Lucs) were transplanted into severe combined immunodeficient mouse hindlimbs to form teratoma. The chemotherapy group was injected with MnCl(2) intraperitoneally three times a week. The control group was given MnCl(2) only prior to manganese-enhanced MRI. Longitudinal evaluation by manganese-enhanced MRI and bioluminescence imaging was performed. The chemotherapy group showed significant reduction in the teratoma volume and luciferase activity at weeks 6 and 8. Histology revealed increased proportion of dead cells and caspase 3 positive cells in the chemotherapy group. Systemic administration of MnCl(2) enabled simultaneous monitoring and elimination of hESC-derived teratoma cells by higher intracellular accumulation of Mn(2+).

The importance of beta cell characterisation: generating human beta cells by differentiating human embryonic stem cells

Exogenous insulin administration continues to be the most common means of correcting the absolute insulin deficiency in patients with type 1 diabetes mellitus. Nevertheless, the best available insulin therapy does not control blood glucose levels in a physiological manner, leading to several serious metabolic and neurovascular complications [1]. Beta cell replacement offers an attractive alternative, potentially enabling precise glycaemic control. Clinical islet transplantation, for instance, has proven effective in improving glucose homeostasis, reducing episodes of hypoglycaemia and achieving insulin independence [2]. However, the shortage of donor islet tissues is a major restriction for widespread application of beta cell replacement therapies. The recent developments in stem cell research suggest that human embryonic stem cells (hESCs) hold great promise for fulfilling this need for an unlimited supply of readily available insulin-producing cells. Embryonic stem cells are pluripotent cells derived from the inner cell mass of blastocysts. They exhibit the capacity to develop into the three embryonic germ layers (endoderm, mesoderm and ectoderm). To generate beta cells from hESCs, current approaches have been guided by knowledge of pancreatic development. By recapitulating these developmental signals, several groups have been able to differentiate hESCs into insulinpositive cells [3–6]. Jiang et al. utilised a differentiation protocol omitting serum from the culture medium and reported the presence of islet-like clusters that secreted Cpeptide in response to glucose [3]. However, C-peptidepositive cells only accounted for 2–8% of all cells in the islet-like cluster [3]. When transplanted into chemically induced diabetic mice, the islet-like clusters prolonged survival and secreted C-peptide in response to a glucose challenge [4]. Novocell (now known as ViaCyte, San Diego, CA, USA) developed another differentiation protocol in which hESC-derived insulin positive cells could secrete C-peptide but only in the presence of secretagogues like KCl [5]. When hESC-derived pancreatic progenitors were transplanted into athymic mice, they reported that these cells could also produce human insulin and C-peptide levels comparable to 3,000 human islets [6]. While the results reported by the ViaCyte group were impressive, Matveyenko et al. had difficulty replicating these results in athymic rats [7]. In addition, the recipient mice responded poorly to glucose tolerance testing, which suggests that insulin secretion was not glucose dependent. Clearly, improvements to directed differentiation attempts are necessary to overcome two significant hurdles that G. S. Korbutt : T. Y. Yeung :C. E. Ellis Department of Surgery, Alberta Diabetes Institute, Edmonton, AB, Canada

INS GFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

Aims/hypothesisWe aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS+) cells derived in vitro.MethodsHomologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS+ cells.ResultsDifferentiation of INS GFP/w hESCs using published protocols demonstrated that all GFP+ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP+ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS GFP/w hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP+ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS+ cells.Conclusions/interpretation INS GFP/w hESCs are a valuable tool for investigating the nature of early INS+ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS+ cells from differentiating hESCs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2379-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Effective Development of Hematopoietic Niche Cells From hESCs: hESC-Derived Stromal Cells Support LTC-ICs Better Than Bone Marrow-Derived Stromal Cells

Abstract 4804Quantification of putative hematopoietic stem cells (HSCs) in human bone marrow or cord blood is typically assessed by its potential to mediate long-term, multilineage engraftment when transplanted into immunodeficient murine recipients. However, in vitro surrogate assays are extremely attractive due to their relative ease of implementation, low cost and improved throughput of results. The long-term culture initiating cell (LTC-IC) assay serves this purpose by quantifying the ability of putative HSCs in a given population to be cultured for an extended period, typically 5 weeks. Previously, we have derived both mesenchymal stromal cells (MSCs) and endothelial cells (ECs) from human embryonic stem cells (hESCs). MSCs and ECs are important components of the osteogenic and vascular hematopoietic niches and hESC-derived stromal cells can provide autologous supporting cell populations for putative hESC-derived HSCs. Therefore, we evaluated if hESC-derived MSCs (hESC-MSCs) and hESC-derived ECs (hESC-ECs) would have improved ability to support LTC-IC activity of UCB and hESC-derived hematopoietic cells compared to MSCs isolated from human bone marrow (BM-MSCs) and a murine stromal line, M2-10B4. In the present study, we utilized LTC-IC culture of both hESC-derived CD34+CD45+ populations and UCB 34+ cells to test their LTC-IC potential on hESC-MSCs, hESC-ECs and a combination of the two hESC-derived populations. LTC-IC frequency and maintenance of input populations was assessed by survival/proliferation and preservation of colony forming cell (CFC) progenitors over 5 weeks. Initial studies showed all stromal layers were able to support short-term (3 week) culture of CFCs from CD34+ UCB. Total cell proliferation was found to be greatest with the hESC-derived stroma at 3 weeks. Additionally, hESC-MSCs and hESC-ECs yielded CFC comparable to M2-10B4 and significantly more than BM-MSCs. Co-culture of CD34+ UCB with a 1:1 mixture of hESC-MSCs and hESC-ECs yielded similar proliferation and CFC results to either stroma alone. After 5 weeks of culture to quantify LTC-ICs, both of the hESC-derived cells again supported expansion of UCB total cell numbers better than BM-MSCs and similar to M2-10B4. LTC-IC yield at 5 weeks was 85 ± 29 and 74 ± 12 per 103 input cells for hESC-MSC and hESC-EC, respectively, as compared to 15 ± 12 for BM-MSCs and 104 ± 56 for M2-10B4 co-culture. Combining the hESC-derived MSCs and ECs produced an LTC-IC yield of 54 ± 4. Next, we tested the ability of hESC-derived CD34+CD45+ cells to produce CFCs and LTC-ICs using co-culture with hESC-MSCs and M2-10B4. In short-term (2-3 week) assays, hESC-derived CD34+CD45+ cells declined in number to about 30% of the starting population on both stroma. However, between 3–5 weeks, cell number remained fairly constant on M2-10B4 while continuing to decrease on hESC-MSC. Interestingly, CFC yield for hESC-derived CD34+CD45+ cells at 2 weeks was 45 ± 43 per 103 input cells on M2-10B4, while co-culture with hESC-MSCs regularly failed to produce any CFC at this time. Between 3–5 weeks of culture no CFC or LTC-ICs were produced from hESC CD34+CD45+ using any of the stromal cells. Similar results were seen for hESC-derived CD34+CD43+ populations. Together, these results have two key findings. First, hESC-derived ECs and MSCs are effective in supporting LTC-ICs from UCB, and do so better than human BM-MSCs. Second, these same stromal cells do not support survival of hESC-derived LTC-ICs. In this manner, these results closely mimic studies that show limited long-term hematopoietic engraftment of hESC-derived cells when transplanted into immunodeficient mice. Therefore, this system can now be used to aid in identification of both hematopoietic niche elements and specific cell populations that mediate development and support of putative HSCs derived from hESCs and iPSCs. Disclosures:No relevant conflicts of interest to declare.

Pluripotent Stem Cell-Derived Human Natural Killer Cells with Potent Anti-Multiple Myeloma Activity,

Abstract 4034Natural Killer (NK) cells are lymphocytes of the innate immune system with anti-viral and anti-cancer activity. Over the past decade, they have gained interest as a promising cellular source for use in adoptive immunotherapy for the treatment of cancer. Most notably, NK cells play an important role in the graft-vs-tumor effect seen in allogeneic hematopoietic stem cell transplantation (allo-HSCT), and a better understanding of NK cell biology has translated into improved transplant outcomes in acute myelogenous leukemia (AML). Small studies have demonstrated a role for NK cell activity in multiple myeloma (MM) patients receiving allo-HSCT. Investigators have also utilized haplo-identical killer immunoglobulin-like receptor (KIR) mismatched NK cells for adoptive immunotherapy in patients with multiple myeloma (MM). Our group has focused on the development of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a novel starting source of lymphocytes for immunotherapy. We have previously demonstrated potent anti-tumor activity of hESC-derived NK cells in vitro and in vivo against a variety of different targets. We have also shown that iPSC-derived NK cells from a variety of different somatic cell starting sources posses potent anti-tumor and anti-viral activity. Here, we demonstrate hESC- and iPSC-derived NK cell development in a completely defined, feeder-free system that is amenable to clinical scale-up. These cultures contain a pure population of mature NK cells devoid of any T or B cell contamination, which are common adverse bystanders of cellular products isolated and enriched from peripheral blood. Our cultures are homogenous for their expression of CD56 and express high levels of effector molecules known to be important in anti-MM activity, including KIR, CD16, NKG2D, NKp46, NKp44, FasL and TRAIL. We have now tested the activity of hESC- and iPSC-derived NK cells against MM tumor cells in order to provide a universal source of lymphocytes for adoptive immunotherapy in patients with treatment refractory disease. We find that similar to peripheral blood NK cells (PB-NK), hESC- and iPSC-derived NK cells are cytotoxic against 3 distinct MM cell lines in a standard chromium release cytotoxicity assay. Specifically, activated PB-NK cells killed 48.5% of targets at 10 to 1 effector to target ratios, whereas hESC (46.3%) and iPSC (42.4%) derived NK cells also demonstrated significant anti-MM activity. Also, hESC- and iPSC-derived NK cells secrete cytokines (IFNγ and TNFα) and degranulate as demonstrated by CD107a surface expression in response to MM target cell stimulation. When tested against freshly isolated samples from MM patients, hESC- and IPSC-derived NK cells respond at a similar level as activated PB-NK cells, the current source of NK cells used in adoptive immunotherapy trials. These MM targets (both cell lines and primary tumor cells) are known to express defined ligands (MICA/B, DR4/5, ULBP-1, BAT3) for receptors expressed on NK cells as well as a number of undefined ligands for natural cytotoxicity receptors (NCRs) and KIR. As these receptor-ligand interactions drive the anti-MM activity of NK cells, we are currently evaluating expression of each of these molecules on the surface of both the effector and target cell populations. Not only do hESC- and iPSC-derived NK cells provide a unique, homogenous cell population to study these interactions, they also provide a genetically tractable source of lymphocytes for improvement of the graft-vs-myeloma effect and could be tailored on a patient specific basis using banks of hESC-or iPSC-derived NK cells with defined KIR genotypes for use as allogeneic or autologous effector cells. Disclosures:No relevant conflicts of interest to declare.

Electroporation Can Efficiently Transfect hESC-Derived Mesenchymal Stem Cells without Inducing Differentiation

Electroporation is a common method of gene transfer that has recently been used to efficiently transfect mesenchymal stem (stromal) cells (MSCs); however, the electrical stimulus has the potential to alter cell state. This study examines possible negative effects of electroporation of human embryonic stem cell (hESC)-derived MSCs including, loss of potency or induction of differentiation. Immunofluorescence and PCR were used to quantify protein and RNA expression of CD73 (an MSC marker) and markers of mature mesenchymal cell types following electroporation. The relative fraction of cells expressing CD73 protein was not altered in cells exposed to a 20 ms pulse at 1000 V or 1500 V compared to controls even after three passages, suggesting MSCs retain multipotency following electroporation. In addition, RNA expression of markers indicative of mature cells of bone, fat and cardiac muscle did not differ from unmanipulated controls soon after electroporation. Taken together, these results indicate electroporation under conditions favorable for MSC transfection does not significantly alter stem cell state.

Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells

Understanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells to defined and functional cell types is essential for future clinical applications. In this study, we have compared transcriptional profiles of haematopoietic progenitors derived from hESCs at various developmental stages of a feeder- and serum-free differentiation method and show that the largest transcriptional changes occur during the first 4 days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11th most upregulated transcript during the first 4 days of the hESC differentiation process. Overexpression of SCL in hESCs promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intrasplenic transplantation of SCL-overexpressing hESC-derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment, suggesting a paracrine-mediated effect.

Large‐scale compatible methods for the preservation of human embryonic stem cells: Current perspectives

Human embryonic stem cells (hESCs) and hESC-derived cells are of great interest, not only because of their therapeutic potential but also their prospective uses in in vitro drug and toxicity screening. The ability to preserve these cells is critical, allowing for the generation of quality-controlled stocks of cells, transport of cells between sites, and avoiding the need for expensive and time-consuming continuous culture. Current methodologies, namely conventional slow freezing and vitrification, can successfully preserve hESCs and their differentiated progeny, retaining the key characteristics of the cells. However, there is a significant gap between the number of cells potentially needed to either treat patients or run a high-throughput drug screen and how many cells can be preserved using these techniques.Therefore, this review focuses on the scalability of slow freezing and vitrification, identifying key barriers to success and whether they can be overcome. Given the precedent with other mammalian cells in using slow freezing to successfully preserve large quantities of cells and its compatibility with current and emerging culture methods for hESCs, it is likely to become the method of choice for cryopreserving these cells at scale. However, issues other than scale still exist; therefore, alternatives to cryopreservation should also be explored. Here,the potential to lyophilize hESCs for long-term storage is considered as one such alternative.

Novel Technique for Inducing Neural Crest Fate in Embryonic Stem Cells (Stem Cells 2009;27:2896-2905)

Hotta et al1 has reported a study entitled Small-molecule induction of neural crest-like cells derived from human neural progenitors in the December 2009 issue of Stem Cells. Neural crest (NC) cells are ectoderm-derived multipotent migratory cells that give rise to diverse cell lineages in a developing embryo. Migratory or proliferative deficit of NC cells results in many developmental disorders. Stem cell-based therapies are potential treatment options for such disorders. The enteric nervous system (ENS) of vertebrates predominantly develops from NC cells originating from the vagal end of the neural tube. In Hirschsprung's disease, the distal bowel lacks an ENS caused by a failure of colonization by NC-derived cells. This study shows a simple method of coculture with mouse embryonic fibroblasts (mEFs) for generating NC-like cells from human embryonic stem cells (hESCs). The authors also demonstrated that subsequent exposure to Y27632, a selective inhibitor of the Rho-associated coiled kinase (ROCK), significantly increased the efficiency of differentiation into NC-like cells. These hESC-derived NC-like cells expressed NC markers including p75 (p75NTR low affinity neurotrophin receptor), SoxE (Sox8, Sox9 and Sox10) and HNK1 (human natural killer-1 antigen). The mEFs induced NC-like cells were also able to migrate along NC pathways in avian embryos and differentiate into cells expressing neuronal and glial markers. The hESC-derived NC-like cells also demonstrated migration in mouse gut explants, however, Y27632 treatment was required for differentiation into neuronal and glial cells.

Microarray Characterization of Human Embryonic Stem Cell–Derived Retinal Cultures

A number of protocols have been published to induce retinal determination from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). Although all these studies have shown some degree of expression of markers of retinal cells, fewer than 30 markers are typically used to characterize the ESC-derived retinal cells. Hence, it is not known whether they express all the genes present in normal developing retinal cells. To assess the efficiency of their retinal determination protocol at the transcriptome level and to understand the changes in human retinal gene expression patterns during development, the authors conducted a microarray-based analysis comparing human retina to hESC-derived retinal cells. The authors extracted total RNA from 60-day, 80-day, and 96-day human fetal retina and hESC-derived retinal cells at 3 weeks and 9 weeks after induction. RNA was subjected to analysis using a commercial microarray. Data were normalized using Affymetrix Power Tools and analyzed using commercial microarray software. On K-median clustering analysis, the authors found that overall there was a very high correlation between genes expressed in human fetal retina and those in ESC-derived retinal cultures. The cultures were at similar developmental ages to the corresponding fetal retinal ages. They found only 1% of the genes on the array to be expressed at a higher level in ESC-derived retinal cells than in fetal retina, and most of these were expressed in the retinal pigment epithelium and ciliary epithelium. In sum, gene array profiling provides an effective method for characterization of the efficiency of directed differentiation of hESCs to retinal cells.

Pancreatic exocrine enzyme-producing cell differentiation via embryoid bodies from human embryonic stem cells

Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all- trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.