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Nature Biotechnology 05/2013; 31(5):416-8. · 29.50 Impact Factor
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ABSTRACT: The efficient generation of hematopoietic stem cells from human pluripotent stem cells is dependent on the appropriate specification of the definitive hematopoietic program during differentiation. In this study, we used T lymphocyte potential to track the onset of definitive hematopoiesis from human embryonic and induced pluripotent stem cells differentiated with specific morphogens in serum- and stromal-free cultures. We show that this program develops from a progenitor population with characteristics of hemogenic endothelium, including the expression of CD34, VE-cadherin, GATA2, LMO2, and RUNX1. Along with T cells, these progenitors display the capacity to generate myeloid and erythroid cells. Manipulation of Activin/Nodal signaling during early stages of differentiation revealed that development of the definitive hematopoietic progenitor population is not dependent on this pathway, distinguishing it from primitive hematopoiesis. Collectively, these findings demonstrate that it is possible to generate T lymphoid progenitors from pluripotent stem cells and that this lineage develops from a population whose emergence marks the onset of human definitive hematopoiesis.
Cell reports. 12/2012;
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ABSTRACT: Primitive erythropoiesis defines the onset of hematopoiesis in the yolk sac of the early embryo and is initiated by the emergence of progenitors assayed as colony-forming cells (EryP-CFCs). EryP-CFCs are detected for only a narrow window during embryonic development, suggesting that both their initiation and termination are tightly controlled. Using the embryonic stem differentiation system to model primitive erythropoiesis, we found that miR-126 regulates the termination of EryP-CFC development. Analyses of miR-126 null embryos revealed that this miR also regulates EryP-CFCs in vivo. We identified vascular cell adhesion molecule-1 (Vcam-1) expressed by a mesenchymal cell population as a relevant target of miR-126. Interaction of EryP-CFCs with Vcam-1 accelerated their maturation to ßh1-globin(+) and Ter119(+) cells through a Src family kinase. These findings uncover a cell nonautonomous regulatory pathway for primitive erythropoiesis that may provide insight into the mechanism(s) controlling the developmental switch from primitive to definitive hematopoiesis.
Developmental cell 06/2012; 23(1):45-57. · 13.36 Impact Factor
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ABSTRACT: The loss of beta cells in Type I diabetes ultimately leads to insulin dependence and major complications that are difficult to manage by insulin injections. Given the complications associated with long-term administration of insulin, cell-replacement therapy is now under consideration as an alternative treatment that may someday provide a cure for this disease. Over the past 10 years, islet transplantation trials have demonstrated that it is possible to replenish beta cell function in Type I diabetes patients and, at least temporarily, eliminate their dependency on insulin. While not yet optimal, the success of these trials has provided proof-of-principle that cell replacement therapy is a viable option for treating diabetes. Limited access to donor islets has launched a search for alternative source of beta cells for cell therapy purposes and focused the efforts of many investigators on the challenge of deriving such cells from human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs). Over the past five years, significant advances have been made in understanding the signaling pathways that control lineage development from human pluripotent stem cells (hPSCs) and as a consequence, it is now possible to routinely generate insulin producing cells from both hESCs and hiPSCs. While these achievements are impressive, significant challenges do still exist, as the majority of insulin producing cells generated under these conditions are polyhormonal and non functional, likely reflecting the emergence of the polyhormonal population that is known to arise in the early embryo during the phase of pancreatic development known as the 'first transition'. Functional beta cells, which arise during the second phase or transition of pancreatic development have been generated from hESCs, however they are detected only following transplantation of progenitor stage cells into immunocompromised mice. With this success, our challenge now is to define the pathways that control the development and maturation of this second transition population from hPSCs, and establish conditions for the generation of functional beta cells in vitro.
Seminars in Cell and Developmental Biology 06/2012; 23(6):701-10. · 6.65 Impact Factor
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ABSTRACT: We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.
Biotechnology and Bioengineering 12/2011; 109(4):853-66. · 3.95 Impact Factor
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ABSTRACT: To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.
Nature Biotechnology 11/2011; 29(11):1011-8. · 29.50 Impact Factor
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Xin Zhang,
Safak Yalcin,
Dung-Fang Lee,
Tsung-Yin J Yeh,
Seung-Min Lee,
Jie Su,
Sathish Kumar Mungamuri,
Pauline Rimmelé,
Marion Kennedy,
Rani Sellers,
Markus Landthaler,
Thomas Tuschl,
Nai-Wen Chi,
Ihor Lemischka, Gordon Keller,
Saghi Ghaffari
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ABSTRACT: Pluripotency of embryonic stem cells (ESCs) is defined by their ability to differentiate into three germ layers and derivative cell types and is established by an interactive network of proteins including OCT4 (also known as POU5F1; ref. 4), NANOG (refs 5, 6), SOX2 (ref. 7) and their binding partners. The forkhead box O (FoxO) transcription factors are evolutionarily conserved regulators of longevity and stress response whose function is inhibited by AKT protein kinase. FoxO proteins are required for the maintenance of somatic and cancer stem cells; however, their function in ESCs is unknown. We show that FOXO1 is essential for the maintenance of human ESC pluripotency, and that an orthologue of FOXO1 (Foxo1) exerts a similar function in mouse ESCs. This function is probably mediated through direct control by FOXO1 of OCT4 and SOX2 gene expression through occupation and activation of their respective promoters. Finally, AKT is not the predominant regulator of FOXO1 in human ESCs. Together these results indicate that FOXO1 is a component of the circuitry of human ESC pluripotency. These findings have critical implications for stem cell biology, development, longevity and reprogramming, with potentially important ramifications for therapy.
Nature Cell Biology 07/2011; 13(9):1092-9. · 19.49 Impact Factor
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M Cristina Nostro,
Farida Sarangi,
Shinichiro Ogawa,
Audrey Holtzinger,
Barbara Corneo,
Xueling Li,
Suzanne J Micallef,
In-Hyun Park,
Christina Basford,
Michael B Wheeler,
George Q Daley,
Andrew G Elefanty,
Edouard G Stanley, Gordon Keller
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ABSTRACT: The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.
Development 03/2011; 138(5):861-71. · 6.60 Impact Factor
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ABSTRACT: Efficient differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to a variety of lineages requires step-wise approaches replicating the key commitment stages found during embryonic development. Here we show that expression of PdgfR-α segregates mouse ESC-derived Flk-1 mesoderm into Flk-1(+)PdgfR-α(+) cardiac and Flk-1(+)PdgfR-α(-) hematopoietic subpopulations. By monitoring Flk-1 and PdgfR-α expression, we found that specification of cardiac mesoderm and cardiomyocytes is determined by remarkably small changes in levels of Activin/Nodal and BMP signaling. Translation to human ESCs and iPSCs revealed that the emergence of cardiac mesoderm could also be monitored by coexpression of KDR and PDGFR-α and that this process was similarly dependent on optimal levels of Activin/Nodal and BMP signaling. Importantly, we found that individual mouse and human pluripotent stem cell lines require optimization of these signaling pathways for efficient cardiac differentiation, illustrating a principle that may well apply in other contexts.
Cell stem cell 02/2011; 8(2):228-40. · 23.56 Impact Factor
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ABSTRACT: Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro, thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells. The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-β and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives.
Nature Biotechnology 02/2011; 29(3):267-72. · 29.50 Impact Factor
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ABSTRACT: In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+) endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.
PLoS ONE 01/2011; 6(9):e24058. · 4.09 Impact Factor
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ABSTRACT: Emerging evidence suggests that myeloid-derived suppressor cells (MDSCs) have great potential as a novel immune intervention modality in the fields of transplantation and autoimmune diseases. Thus far, efforts to develop MDSC-based therapeutic strategies have been hampered by the lack of a reliable source of MDSCs. Here we show that functional MDSCs can be efficiently generated from mouse embryonic stem (ES) cells and bone marrow hematopoietic stem (HS) cells. In vitro-derived MDSCs encompass two homogenous subpopulations: CD115(+)Ly-6C(+) and CD115(+)Ly-6C(-) cells. The CD115(+)Ly-6C(+) subset is equivalent to the monocytic Gr-1(+)CD115(+)F4/80(+) MDSCs found in tumor-bearing mice. In contrast, the CD115(+)Ly-6C(-) cells, a previously unreported population of MDSCs, resemble the granulocyte/macrophage progenitors developmentally. In vitro, ES- and HS-MDSCs exhibit robust suppression against T-cell proliferation induced by polyclonal stimuli or alloantigens via multiple mechanisms involving nitric oxide synthase-mediated NO production and interleukin (IL)-10. Impressively, they display even stronger suppressive activity and significantly enhance ability to induce CD4(+)CD25(+)Foxp3(+) regulatory T-cell development compared with tumor-derived MDSCs. Furthermore, adoptive transfer of ES-MDSCs can effectively prevent alloreactive T-cell-mediated lethal graft-versus-host disease, leading to nearly 82% long-term survival among treated mice. The successful in vitro generation of MDSCs may represent a critical step toward potential clinical application of MDSCs.
Stem Cells 03/2010; 28(3):620-32. · 7.78 Impact Factor
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Atsushi Kubo,
Yon Hui Kim,
Stefan Irion,
Shogo Kasuda,
Mitsuaki Takeuchi,
Kazuo Ohashi,
Masayuki Iwano,
Yoshiko Dohi,
Yoshihiko Saito,
Ralph Snodgrass, Gordon Keller
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ABSTRACT: We investigated the role of the hematopoietically expressed homeobox (Hex) in the differentiation and development of hepatocytes within embryonic stem cell (ESC)-derived embryoid bodies (EBs). Analyses of hepatic endoderm derived from Hex(-/-) EBs revealed a dramatic reduction in the levels of albumin (Alb) and alpha-fetoprotein (Afp) expression. In contrast, stage-specific forced expression of Hex in EBs from wild-type ESCs led to the up-regulation of Alb and Afp expression and secretion of Alb and transferrin. These inductive effects were restricted to c-kit(+) endoderm-enriched EB-derived populations, suggesting that Hex functions at the level of hepatic specification of endoderm in this model. Microarray analysis revealed that Hex regulated the expression of a broad spectrum of hepatocyte-related genes, including fibrinogens, apolipoproteins, and cytochromes. When added to the endoderm-induced EBs, bone morphogenetic protein 4 acted synergistically with Hex in the induction of expression of Alb, Afp, carbamoyl phosphate synthetase, transcription factor 1, and CCAAT/enhancer binding protein alpha. These findings indicate that Hex plays a pivotal role during induction of liver development from endoderm in this in vitro model and suggest that this strategy may provide important insight into the generation of functional hepatocytes from ESCs.
Hepatology 02/2010; 51(2):633-41. · 11.66 Impact Factor
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Eric D Adler,
Vincent C Chen,
Anne Bystrup,
Aaron D Kaplan,
Steven Giovannone,
Karen Briley-Saebo,
Wilson Young,
Steve Kattman,
Venkatesh Mani,
Michael Laflamme,
Wei-Zhong Zhu,
Zahi Fayad, Gordon Keller
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ABSTRACT: We recently described a murine embryonic stem cell (ESC) line engineered to express the activated Notch 4 receptor in a tetracycline (doxcycline; Dox) regulated fashion (tet-notch4 ESCs). Notch 4 induction in Flk1(+) hematopoietic and vascular progenitors from this line respecified them to a cardiovascular fate. We reasoned that these cells would be ideal for evaluating the contribution of the cardiomyocyte and vascular lineages to the functional improvement noted following stem cell transplantation in infarcted hearts. Flk-1(+) Tet-notch4 cells from d 3 embryoid bodies exposed to doxycycline (Dox(+)) were compared to uninduced (Dox(-)) Flk-1(+) cells. Mice underwent transplantation of 5 x 10(5) Dox(+) cells, Dox(-)cells, or an equal volume of serum-free medium after surgically induced myocardial infarction. The mean ejection fraction was 59 + or - 15, 46 + or - 17, and 39 + or - 13% in the Dox(+), Dox(-), and serum-free medium groups, respectively (P<0.05 for the differences among all 3 groups). Immunohistochemistry of hearts injected with Dox(+) grafts expressed myocardial and vascular markers, whereas grafts of Dox(-) cells expressed primarily vascular markers. We conclude that cardiovascular progenitors are more effective than vascular progenitors in improving function after myocardial infarction. The transplantation of appropriate cell types is critical for maximizing the benefit of cardiovascular cell therapy.-Adler, E. D., Chen, V. C., Bystrup, A., Kaplan, A. D., Giovannone, S., Briley-Saebo, K., Young, W., Kattman, S., Mani, V., Laflamme, M., Zhu, W.-Z., Fayad, Z., Keller, G. The cardiomyocyte lineage is critical for optimization of stem cell therapy in a mouse model of myocardial infarction.
The FASEB Journal 11/2009; 24(4):1073-81. · 5.71 Impact Factor
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Cell stem cell 04/2009; 4(3):198-9; author reply 202. · 23.56 Impact Factor
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ABSTRACT: To efficiently generate cardiomyocytes from embryonic stem (ES) cells in culture it is essential to identify key regulators of the cardiac lineage and to develop methods to control them. Using a tet-inducible mouse ES cell line to enforce expression of a constitutively activated form of the Notch 4 receptor, we show that signaling through the Notch pathway can efficiently respecify hemangioblasts to a cardiac fate, resulting in the generation of populations consisting of >60% cardiomyocytes. Microarray analyses reveal that this respecification is mediated in part through the coordinated regulation of the BMP and Wnt pathways by Notch signaling. Together, these findings have uncovered a potential role for the Notch pathway in cardiac development and provide an approach for generating large numbers of cardiac progenitors from ES cells.
Nature Biotechnology 10/2008; 26(10):1169-78. · 29.50 Impact Factor
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ABSTRACT: Patients with glioblastomas have a poor prognosis and new treatments paradigms are needed. The futility of current treatments lies in part on the difficulty of delivering therapeutic agents to the tumor. In this article, we review and discuss current knowledge of adjuvant treatment for glioblastomas including gene therapy and viral vectors. Additionally, this review provides an update on recent progress, which has focused on improving delivery methods including the development of new approaches such as stem cells. Particular emphasis is given to laboratory data showing the use of embryonic stem cells used as vectors to deliver pro-apoptotic genes to glioblastoma cells.
Cancer biology & therapy 10/2008; 7(9):1341-7. · 2.64 Impact Factor
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ABSTRACT: The treatment of malignant gliomas with current therapies remains a challenge in neurooncology. Our recent work showed that embryonic stem cell (ESC)-derived astrocytes conditionally expressing genes can be used to induce apoptosis in malignant glioma cells in vitro. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene has been shown to induce apoptosis in a variety of tumor cells, including gliomas. The aim of this study was to assess the proapoptotic effects of transgenic TRAIL delivered by ESC-derived astrocytes on malignant gliomas in vivo. Malignant glioma A172 cells were used to induce heterotopic xenografts in nude mice. ESC-derived astrocytes conditionally expressing TRAIL were injected into the xenografts. TRAIL expression was documented in the malignant glioma xenografts by reverse transcription PCR and immunohistochemistry after external gene induction. A significant reduction in tumor volume occurred 48 h after a single injection (14%) and double injections (31%) in the experimental groups. Terminal dUTP nick end labeling (TUNEL) revealed abundant apoptotic tumor cells in the experimental groups. Seven days after injection, the tumor had undergone severe necrosis, with only scattered residual tumor cells at the periphery. Death receptor DR4 expression increased significantly in the experimental groups compared with controls. Our data suggest that ESC-derived astrocytes conditionally expressing TRAIL should be considered as vectors to deliver gene therapy for malignant gliomas.
Neuro-Oncology 09/2008; 11(2):102-8. · 5.72 Impact Factor
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ABSTRACT: Myocardial regeneration with stem-cell transplantation is a possible treatment option to reverse deleterious effects that occur after myocardial infarction. Since little is known about stem cell survival after transplantation, developing techniques for "tracking" cells would be desirable. Iron-oxide-labeled stem cells have been used for in vivo tracking using MRI but produce negative contrast images that are difficult to interpret. The aim of the current study was to test a positive contrast MR technique using reduced z-gradient rephasing (GRASP) to aid in dynamically tracking stem cells in an in vivo model of mouse myocardial infraction. Ferumoxides and protamine sulfate were complexed and used to magnetically label embryonic stem cell-derived cardiac-precursor-cells (ES-CPCs). A total of 500,000 ES-CPCs were injected in the border zone of infarcted mice and MR imaging was performed on a 9.4T scanner using T(2)*-GRE sequences (negative contrast) and positive contrast GRASP technique before, 24 hours, and 1 week after ES-CPC implantation. Following imaging, mice were sacrificed for histology and Perl's staining was used to confirm iron within myocardium. Good correlation was observed between signal loss seen on conventional T(2)* images, bright areas on GRASP, and the presence of iron on histology. This demonstrated the feasibility of in vivo stem cell imaging with positive contrast MRI.
Magnetic Resonance in Medicine 08/2008; 60(1):73-81. · 2.96 Impact Factor
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ABSTRACT: The derivation of human embryonic stem (hES) cells has opened new avenues for studies on human development and provided a potential source of cells for replacement therapy. To reveal the full potential of hES cells, it would be advantageous to be able to genetically alter them as is routinely done with mouse ES cells through homologous recombination. The mouse Rosa26 locus is particularly useful for genetic modification as it can be targeted with high efficiency and is expressed in most cell types tested. Here we report the identification of the human homolog of the mouse Rosa26 locus. We demonstrate targeting of a red-fluorescent protein (tdRFP) cDNA to this locus through homologous recombination and expression of this targeted reporter in multiple hES cell-derived lineages. Through recombinase-mediated cassette exchange, we show replacement of the tdRFP cDNA with other cDNAs, providing a cell line in which transgenes can be readily introduced into a broadly expressed locus.
Nature Biotechnology 01/2008; 25(12):1477-82. · 29.50 Impact Factor
