hESC-derived Cells Research Articles

Differentiation of Human Embryonic Stem Cell–Derived Retinal Progenitors into Retinal Cells by Sonic Hedgehog and/or Retinal Pigmented Epithelium and Transplantation into the Subretinal Space of Sodium Iodate–Injected Rabbits

Transplantation of retinal cells has recently provided a promising therapeutic approach for retinal degeneration. Here, we differentiated initially retinal progenitors (RPs) from adherent feeder-free human embryonic stem cells (hESCs) with the use of defined media supplemented with a specific combination of growth factors. The differentiated RPs highly (>80%) expressed related molecular features that included Six3 at an early stage in addition to Crx, Rx, Pax6, Otx2, and Chx10 at later stage. Next, we examined the induction of photoreceptors by Shh and/or the coculture of rabbit retinal pigmented epithelium with hESCs-derived RPs. The differentiation of retinal cells was demonstrated by protein and gene expression in all groups. However, S-Opsin, a cone photoreceptor marker, had higher expression in the presence of Shh, whereas expressions of Gli and Hes1 decreased in the same group. Finally, hESC-derived RPs were treated with Shh transplanted into the subretinal space of sodium iodate-injected albino-type adult rabbits and analyzed 4 weeks later. Transplanted retinal cells survived, migrated into retinal layers, and restored a small but significant B-wave. The grafted cells expressed photoreceptor markers, S-Opsin and Rhodopsin. Our results indicate that putative hESC-derived retinal cells express related genes and proteins. Further, our results show that retinal-like cells can be useful replacements for photoreceptors in retinal diseases.

The promise of human embryonic stem cells in aging-associated diseases

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications.

Toward Preclinical Predictive Drug Testing for Metabolism and Hepatotoxicity by Using In Vitro Models Derived from Human Embryonic Stem Cells and Human Cell Lines — A Report on the Vitrocellomics EU-project

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.

Microvascular Mural Cell Functionality of Human Embryonic Stem Cell-Derived Mesenchymal Cells

Microvascular mural or perivascular cells are required for the stabilization and maturation of the remodeling vasculature. However, much less is known about their biology and function compared to large vessel smooth muscle cells. We have developed lines of multipotent mesenchymal cells from human embryonic stem cells (hES-MC); we hypothesize that these can function as perivascular mural cells. Here we show that the derived cells do not form teratomas in SCID mice and independently derived lines show similar patterns of gene expression by microarray analysis. When exposed to platelet-derived growth factor-BB, the platelet-derived growth factor receptor β is activated and hES-MC migrate in response to a gradient. We also show that in a serum-free medium, transforming growth factor β1 (TGFβ1) induces robust expression of multiple contractile proteins (α smooth muscle actin, smooth muscle myosin heavy chain, smooth muscle 22α, and calponin). TGFβ1 signaling is mediated through the TGFβR1/Alk5 pathway as demonstrated by inhibition of α smooth muscle actin expression by treatment of the Alk5-specific inhibitor SB525334 and stable retroviral expression of the Alk5 dominant negative (K232R). Coculture of human umbilical vein endothelial cell (HUVEC) with hES-MC maintains network integrity compared to HUVEC alone in three-dimensional collagen I-fibronectin by paracrine signaling. Using high-resolution laser confocal microscopy, we show that hES-MC also make direct contact with HUVEC. This demonstrates that hESC-derived mesenchymal cells possess the molecular machinery expected in a perivascular progenitor cells and can play a functional role in stabilizing EC networks in in vitro three-dimensional culture.

Functional Characterization and Expression Profiling of Human Induced Pluripotent Stem Cell- and Embryonic Stem Cell-Derived Endothelial Cells

With regard to human induced pluripotent stem cells (hiPSCs), in which adult cells are reprogrammed into embryonic-like cells using defined factors, their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study, hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method, and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the "distances" among hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and HUVECs. We further conclude that hiPSCs can differentiate into functional endothelial cells, but with limited expansion potential compared with hESC-ECs; thus, extensive studies should be performed to explore the cause and extent of such differences before clinical application of hiPSC-ECs can begin.

Cardiotoxicity testing using pluripotent stem cell‐derived human cardiomyocytes and state‐of‐the‐art bioanalytics: a review

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given.

Hepatic Differentiation of Human Embryonic Stem Cells Is Promoted by Three-Dimensional Dynamic Perfusion Culture Conditions

The developmental potential of human embryonic stem cells (hESCs) holds great promise to provide a source of human hepatocytes for use in drug discovery, toxicology, hepatitis research, and extracorporeal bioartificial liver support. There are, however, limitations to induce fully functional hepatocytes on conventional two-dimensional (2D) static culture. It had been shown that dynamic three-dimensional (3D) perfusion culture is superior to induce maturation in fetal hepatocytes and prolong hepatic functions of primary adult hepatocytes. We investigated the potential of using a four-compartment 3D perfusion culture to induce hepatic differentiation in hESC. Undifferentiated hESC were inoculated into hollow fiber-based 3D perfusion bioreactors with integral oxygenation. Hepatic differentiation was induced with a multistep growth factor cocktail protocol. Parallel controls were operated under equal perfusion conditions without the growth factor supplementations to allow for spontaneous differentiation, as well as in conventional 2D static conditions using growth factors. Metabolism, hepatocyte-specific gene expression, protein expression, and hepatic function were evaluated after 20 days. Significantly upregulated hepatic gene expression was observed in the hepatic differentiation 3D culture group. Ammonia metabolism activity and albumin production was observed in the 3D directed differentiation culture. Drug-induced cytochrome P450 gene expression was increased with rifampicin induction. Using flow cytometry analysis the mature hepatocyte marker asialoglycoprotein receptor was found on up to 30% of the cells in the 3D system with directed hepatic differentiation. Histological and immunohistochemical analysis revealed structural formation of hepatic and biliary marker-positive cells. In contrast to 2D culture, the 3D perfusion culture induced more functional maturation in hESC-derived hepatic cells. 3D perfusion bioreactor technologies may be useful for further studies on generating hESC-derived hepatic cells.

A Fibrin Patch-Based Enhanced Delivery of Human Embryonic Stem Cell-Derived Vascular Cell Transplantation in a Porcine Model of Postinfarction Left Ventricular Remodeling

It is unknown how to use human embryonic stem cell (hESC) to effectively treat hearts with postinfarction left ventricular (LV) remodeling. Using a porcine model of postinfarction LV remodeling, this study examined the functional improvement of enhanced delivery of combined transplantation of hESC-derived endothelial cells (ECs) and hESC-derived smooth muscle cells (SMCs) with a fibrin three-dimensional (3D) porous scaffold biomatrix. To facilitate tracking the transplanted cells, the hESCs were genetically modified to stably express green fluorescent protein and luciferase (GFP/Luc). Myocardial infarction (MI) was created by ligating the first diagonal coronary artery for 60 minutes followed by reperfusion. Two million each of GFP/Luc hESC-derived ECs and SMCs were seeded in the 3D porous biomatrix patch and applied to the region of ischemia/reperfusion for cell group (MI+P+C, n = 6), whereas biomatrix without cell (MI+P, n = 5), or saline only (MI, n = 5) were applied to control group hearts with same coronary artery ligation. Functional outcome (1 and 4 weeks follow-up) of stem cell transplantation was assessed by cardiac magnetic resonance imaging. The transplantation of hESC-derived vascular cells resulted in significant LV functional improvement. Significant engraftment of hESC-derived cells was confirmed by both in vivo and ex vivo bioluminescent imaging. The mechanism underlying the functional beneficial effects of cardiac progenitor transplantation is attributed to the increased neovascularization. These findings demonstrate a promising therapeutic potential of using these hESC-derived vascular cell types and the mode of patch delivery.

Expression profiles of miRNAs in human embryonic stem cells during hepatocyte differentiation

Human embryonic stem cells (hESCs) are able to self-renew and differentiate into a variety of cell types. Although miRNAs have emerged as key regulators in the cellular process, a few studies have been reported about behaviors of miRNAs during differentiation of hESCs into a specialized cell type. Here, we demonstrate that different kinds of miRNAs may function in a lineage-specific manner during the differentiation of human embryonic stem cells (hESCs). hESCs were induced to definitive endoderm (DE) cells and further differentiated to hepatocytes. The expression levels of miRNAs were examined in hESCs, DE cells, and hepatocytes by miRNA array using 799 human miRNA probes. Among 387 miRNAs significantly detected, 13 and 56 miRNAs were downregulated and upregulated during transition of hESCs to DE cells, respectively, while 30 and 92 miRNAs were downregulated and upregulated during differentiation of DE cells to hepatocytes, respectively. In particular, 5, 4, and 86 miRNAs were enriched in hESCs, DE cells, and hepatocytes, respectively. Quantitative RT-PCR represented that miR-512-3p, miR-512-5p and miR-520c-3p were enriched in hESCs, miR-9*, miR-205 and miR-375 in hESC-derived DE cells, and miR-10a, miR-122 and miR-21 in hESC-derived hepatocytes. Expression patterns of lineage-specific miRNAs in the liver tissue were similar to those of hESC-derived hepatocytes. The results indicate that different kinds of miRNAs may function in a lineage-specific manner during differentiation of hESCs into a specialized cell type.

Historical Perspective: Beginnings of the β-Cell

Over the course of the last half-century, we have gleaned much about the developmental biology of the pancreas and in particular the insulin-producing β-cell, the autoimmune destruction of which results in type 1 diabetes. Deciphering the mechanisms driving β-cell neogenesis in vivo holds great allure, in large part because of the potential therapeutic applications that stand to be gained. The field of pancreas development was arguably established by such scientists as Rutter, Grobstein, Wessells, and Cohen in the 1960s. Their seminal studies were the first to demonstrate the significance of the mesenchyme in supporting development of the pancreas or indeed any organ. Their work was further extended by Teitelman and colleagues who did much to characterize pancreatic differentiation via immunohistochemistry. Over the last two decades, the emergence of increasingly elaborate knockout and transgenic mouse technologies has exponentially expanded our insight into the signaling pathways and transcriptional nexus governing pancreas development. Thus, it is fitting that this review will mainly focus on the development of the pancreas and β-cells in the mouse. Through recapitulating endogenous signaling pathways governing β-cell neogenesis in the embryo, it has recently proven possible to generate insulin-producing cells in vitro from human embryonic stem cells (hESCs) (1). Although this milestone accentuates the great therapeutic potential of studying β-cell neogenesis in vivo, the currently insufficient functionality of hESC-derived insulin cells argues the case for further examination of β-cell development in order to understand how and why such engineered cells differ from their endogenous counterparts. It is likely that resolving these differences will lie in better characterizing the relationships between the many signaling pathways and key factors already known to govern the pancreatic program in regards to spatial and temporal pancreatic expression and their impact on pancreatic differentiation. It is envisaged that such incremental knowledge gains will be applied …

A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells

Here, we have investigated mitochondrial biology and energy metabolism in human embryonic stem cells (hESCs) and hESC-derived neural stem cells (NSCs). Although stem cells collectively in vivo might be expected to rely primarily on anaerobic glycolysis for ATP supply, to minimise production of reactive oxygen species, we show that in vitro this is not so: hESCs generate an estimated 77% of their ATP through oxidative phosphorylation. Upon differentiation of hESCs into NSCs, oxidative phosphorylation declines both in absolute rate and in importance relative to glycolysis. A bias towards ATP supply from oxidative phosphorylation in hESCs is consistent with the expression levels of the mitochondrial gene regulators peroxisome-proliferator-activated receptor γ coactivator (PGC)-1α, PGC-1β and receptor-interacting protein 140 (RIP140) in hESCs when compared with a panel of differentiated cell types. Analysis of the ATP demand showed that the slower ATP turnover in NSCs was associated with a slower rate of most energy-demanding processes but occurred without a reduction in the cellular growth rate. This mismatch is probably explained by a higher rate of macromolecule secretion in hESCs, on the basis of evidence from electron microscopy and an analysis of conditioned media. Taken together, our developmental model provides an understanding of the metabolic transition from hESCs to more quiescent somatic cell types, and supports important roles for mitochondria and secretion in hESC biology.

Identification and Characterization of Adenovirus Early Region 1B-Associated Protein 5 as a Surface Marker on Undifferentiated Human Embryonic Stem Cells

Pluripotent human embryonic stem cells (hESCs) provide appropriate systems for developmental studies and prospective donor cell sources for regenerative medicine. Identification of surface markers specific to hESCs is a prerequisite for studying hESC biology and can be used to generate clinical-level donor cell preparations that are free from tumorigenic undifferentiated hESCs. We previously reported the generation of monoclonal antibodies that specifically recognize hESC surface antigens using a decoy immunization strategy. In this study, we show that monoclonal antibody 57-C11 recognizes a phosphorylated form of adenovirus early region 1B-associated protein 5 (E1B-AP5). E1B-AP5 is a nuclear RNA-binding protein, but we report that 57-C11-reactive E1B-AP5 is expressed on the surface of undifferentiated hESCs. In undifferentiated hESCs, 57-C11-reactive E1B-AP5 is localized to SSEA3-, SSEA4-, TRA-1-60-, TRA-1-81-, OCT4-, SOX2-, and NANOG-positive hESCs. In mixtures of undifferentiated hESCs and hESC-derived neurons, 57-C11 exclusively recognizes undifferentiated hESCs but not hESC-derived neuronal cells. Further, the expression of 57-C11-reactive E1B-AP5 decreases upon differentiation. Our results demonstrate that 57-C11-reactive E1B-AP5 is a novel surface molecule that is involved in the undifferentiated state of hESCs. As far as we know, this is the first report demonstrating that heterogeneous nuclear RNA-binding protein is expressed on the surface of undifferentiated hESCs.

Potential Clinical Applications for Human Pluripotent Stem Cell-Derived Blood Components

The ability of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to divide indefinitely without losing pluripotency and to theoretically differentiate into any cell type in the body makes them highly attractive cell sources for large scale regenerative medicine purposes. The current use of adult stem cell-derived products in hematologic intervention sets an important precedent and provides a guide for developing hESC/iPSC based therapies for the blood system. In this review, we highlight biological functions of mature cells of the blood, clinical conditions requiring the transfusion or stimulation of these cells, and the potential for hESC/iPSC-derivatives to serve as functional replacements. Many researchers have already been able to differentiate hESCs and/or iPSCs into specific mature blood cell types. For example, hESC-derived red blood cells and platelets are functional in tasks such as oxygen delivery and blood clotting, respectively and may be able to serve as substitutes for their donor-derived counterparts in emergencies. hESC-derived dendritic cells are functional in antigen-presentation and may be used as off-the-shelf vaccine therapies to stimulate antigen-specific immune responses against cancer cells. However, in vitro differentiation systems used to generate these cells will need further optimization before hESC/iPSC-derived blood components can be used clinically.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1low, BMPR1Blow, FLT4low, LRRC32low, and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

Hepatic differentiation of human embryonic stem cells in a 3D bioreactor environment

A number of articles addressing the hepatic differentiation of human embryonic stem cells (hESC) in conventional 2D culture systems have been published recently. However, the cell numbers and purity available through this culture method are limited. This study aims to investigate the hepatic differentiation of hESC in a dynamic 3D culture environment that supports cell differentiation and three-dimensional tissue reorganisation in vitro. Based on a perfused 3D-multicompartment bioreactor technology successfully used for primary human liver cell culture, a downscaled system (0.5ml cell volume) was developed to investigate the differentiation, maturation and function of the hESC-derived hepatic cells in vitro. Undifferentiated hESC were inoculated into a miniaturized 3D bioreactor and hepatic differentiation was induced by the sequential application of growth factors, hormones and cytokines. Metabolic parameters indicating metabolic activities (glucose, lactate) of the cells were measured daily. After 30 days the bioreactor was opened and samples for immunofluorescence staining and RNA analysis were taken.The time courses of glucose and lactate indicated stable cell metabolism over 30 days. Immunofluorescence analysis showed the expression of the biliary marker cytokeratin 19 (CK19), and CK18 as hepatocytic / biliary marker indicating hepatic differentiation. Both markers were localized predominantly at the peripheral areas of cell aggregates. These findings are consistent with the detected induction in the expression of albumin, AFP and cytochrome P450 2D6 in samples taken from the bioreactor following hepatic maturation of hESC compared to undifferentiated hESC. In conclusion, the results of the study suggest that the 3D-multicompartment bioreactor supports hepatic differentiation of hESC. Further studies are needed to increase the yield and maturation degree of the cells.

Dysregulated Gene Expression During Hematopoietic Differentiation From Human Embryonic Stem Cells

The generation of hematopoietic cells from human embryonic stem cells (hESC) has raised the possibility of using hESC as an alternative donor source for transplantation. However, functional defects identified in hESC-derived cells limit their use for full lymphohematopoietic reconstitution. The purpose of the present study was to define and quantitate key functional and molecular differences between CD34(+) hematopoietic progenitor subsets derived from hESC and CD34(+) subsets from umbilical cord blood (UCB) representing definitive hematopoiesis. Two distinct sub-populations were generated following mesodermal differentiation from hESC, a CD34(bright) (hematoendothelial) and CD34(dim) (hematopoietic-restricted) subset. Limiting dilution analysis revealed profound defects in clonal proliferation relative to UCB particularly in B lymphoid conditions. Transcription factors normally expressed at specific commitment stages during B lymphoid development from UCB-CD34(+) cells were aberrantly expressed in hESC-derived CD34(+) cells. Moreover, strong negative regulators of lymphopoiesis such as the adaptor protein LNK and CCAAT/enhancer-binding protein-α (CEBPα), were exclusively expressed in hESC-CD34(+) subsets. Knockdown of LNK lead to an increase in hematopoietic progenitors generated from hESCs. The aberrant molecular profile seen in hESC-CD34(+) cells represents persistence of transcripts first expressed in undifferentiated hESC and/or CD326-CD56(+) mesoderm progenitors, and may contribute to the block in definitive hematopoiesis from hESC.

Corning® Synthemax™ Surface: a tool for feeder-free, xeno-free culture of human embryonic stem cells

Currently, human embryonic stem cells (hESCs) are cultured on feeder cells or complex mixtures of proteins extracted from mouse tumors1,2. To allow commercialization of hESC-derived therapeutic cells, culture methods are required that are robust and scalable and that use chemically defined, xeno-free materials. Corning Synthemax Surface is a novel synthetic surface that permits consistent long-term self-renewal of multiple hESC lines in defined, xeno-free media and differentiation of cells to functional cardiomyocytes3.

Hematopoietic differentiation from human ESCs as a model for developmental studies and future clinical translations. Invited review following the FEBS Anniversary Prize received on 5 July 2009 at the 34th FEBS Congress in Prague

Human embryonic stem cells (hESCs) and induced pluripotent stem cells are excellent models for the study of embryonic hematopoiesis in vitro, aiding the design of new differentiation models that may be applicable to cell-replacement therapies. Adult and fetal hematopoietic stem cells are currently being used in biomedical applications; however, the latest advances in regenerative medicine and stem cell biology suggest that hESC-derived hematopoietic stem cells are an outstanding tool for enhancing immunotherapy and treatments for blood disorders and cancer, for example. In this review, we compare various methods used for inducing in vitro hematopoietic differentiation from hESCs, based on co-culture with stromal cells or formation of embryoid bodies, and analyse their ability to give rise to hematopoietic precursors, with emphasis on their engraftment potential as a measure of their functionality in vivo.

Differentiation of Human Natural Killer Cells From Pluripotent Stem Cells In a Feeder Free System Amenable to Clinical Translation

AbstractAbstract 107Human natural killer (NK) cells are an attractive source of lymphocytes for adoptive immunotherapy. Although our understanding of natural killer cell biology continually grows, translating these concepts to the clinic has fallen behind. Currently, NK cell adoptive immunotherapy is only beneficial to a limited number of patients, particularly those with acute myelogenous leukemia (AML). In order to generate NK cells that are effective against a broader range of malignancies, our lab has focused on the generation of NK cells from human pluripotent stem cells. We have previously demonstrated the potency of NK cells derived from human embryonic stem cells (hESCs) both in vitro and in vivo. These previous studies utilized a stromal cell co-culture system to derive hematopoietic progenitor cells (CD34+CD45+ cells) from hESCs that can then produce CD45+CD56+ NK cells in a secondary culture system. More recently, we used a similar co-culture system to generate fully functional NK cells from several human induced pluripotent stem cell (iPSC) lines, which could provide NK cells on a patient-specific basis. Both hESC and iPSC-derived NK cells consist of a mature, homogenous population of cells expressing CD56, CD94, killer immunoglobulin-like receptors (KIRs), CD16, and the apoptosis-inducing ligands FasL and TRAIL. We now aim to convert this system into a completely defined stromal cell-free system. These studies will make this system more amenable to clinical scale up and allow us to better define elements essential for hematopoietic and NK cell development from human pluripotent stem cells. For example, hESC-derived NK cells would require minimal cell processing compared peripheral blood NK (PB-NK) cells that typically requires depletion of CD3+ and CD20+ cells to prevent GVH and passenger lymphocyte syndrome, respectively. Neither T cells nor B cells are present in our cultures, preventing these complications. Here, we have now used a spin embryoid body (“spin-EB”) approach potentially suitable for clinical scale-up. In these spin-EB cultures, defined numbers of undifferentiated hESCs or iPSCs are first aggregated in 96 well plates by centrifugation (3000 cells per well) in serum-free media containing only the cytokines SCF, BMP4, and VEGF. Under these stage 1 conditions, hematopoietic progenitor cells that express CD34, CD45, CD43, and CD31 develop and expand over 6–11 days. After this time, EBs are directly transferred (without dissociation or sorting) to Stage 2 cultures with EL08 stromal cells or stroma-free conditions in serum-free media containing NK cell-initiating cytokines (IL15, SCF, FLT3L, IL7, and IL3). We find that these Stage 2 cells acquire all the typical markers of mature NK cells (CD56, CD94, KIRs, etc). They also kill the CML target K562 cells at similar effector-to-target ratios as stromal-derived NK cells and PB-NK cells. Most notably, spin-EB-derived NK cells generated in entirely feeder free conditions also exhibit a typical NK cell phenotype and kill K562 targets. However, the NK cells derived in feeder-free Stage 2 conditions are less proliferative than those cultured in Stage 2 conditions on EL08 cells. Specifically, 12,000 undifferentiated hESCs or iPSCs give rise to 0.8–1 × 106 NK cells using EL08 in Stage 2 cultures compared to 0.4– 1 × 105 cells using the feeder-free Stage 2 conditions. Additionally, the efficiency of NK cell development from spin-EB cultures is not uniform: 58% of EBs produce NK cells in Stage 2 using EL08 stromal cells, and 25% of EBs produce NK cells in the completely feeder-free system. Based on these calculations, given 100% efficiency of this spin-EB NK cell developmental system, we estimate that 240,000 starting hESCs would provide enough NK cells to treat a single patient with a dose of 20 × 106 NK cells. Even at the lower efficiencies demonstrated here, one plate of undifferentiated hESCs or iPSCs (typically with 5–10 × 106 undifferentiated cells) could treat several patients. Ongoing work is focused on enhancing the percentage of hematopoietic progenitors obtained from the spin EB approach and expanding feeder-free NK cells with artificial antigen presenting cells in Stage 2 conditions. Disclosures:No relevant conflicts of interest to declare.

Methotrexate supports in vivo selection of human embryonic stem cell derived-hematopoietic cells expressing dihydrofolate reductase

Human embryonic stem cells (hESCs) are an attractive alternative cell source for hematopoietic gene therapy applications as the cells are easily modified with lentiviral or other vectors and can be subsequently induced to differentiate into hematopoietic progenitor cells. However, demonstration of the full hematopoietic potential of hESC-derived progeny is challenging due to low marrow engraftment and the difficulty of detecting cells in the peripheral blood of human/mouse xenografts. Methotrexate (MTX) chemotherapy coupled with expression of a drug resistant dihydrofolate reductase such as Tyr22 (Tyr22DHFR) has the potential to selectively increase engraftment of gene-modified human hematopoietic cells in mice, which would allow for better phenotypic characterization of hESC-derived cells in vivo. We showed that hESCs transduced with Tyr22DHFR-GFP encoding lentivirus vectors differentiate into MTX resistant (MTXr) hemato-endothelial cells. MTX treatment of immunodeficient mice infused with Tyr22DHFR hESC-derived hemato-endothelial cells increased the long-term engraftment of human cells in the bone marrow of MTX-treated mice. In contrast to previous studies, these results indicate that MTX administration has the potential to support in vivo selection that is maintained after cessation of treatment. The MTX/Tyr22DHFR system may therefore be useful for enrichment of gene-modified cell populations in human stem cell and gene therapy applications.