Targeting Pancreatic Progenitor Cells in Human Embryonic Stem Cell Differentiation for the Identification of Novel Cell Surface Markers

New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.     

Role of BMP Signaling in Pancreatic Progenitor Differentiation from Human Embryonic Stem Cells

Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.     

利用Tet-On系统构建稳定表达Pdx1的小鼠ESC细胞株

目的:利用Tet-On系统构建稳定表达胰十二指肠同源异形框1(Pdx1)的小鼠胚胎干细胞(ESC)株,为进一步研究Pdx1+定型内胚层细胞向胰腺细胞分化奠定了基础.方法:采用Tet-On系统构建具有绿色荧光蛋白标记及嘌呤霉素抗性的Pdx1过表达慢病毒载体并感染胚胎干细胞.实验分为空白对照组(ESC组)、空载慢病毒对照组...     

Tal1/Scl gene transduction using a lentiviral vector stimulates highly efficient hematopoietic cell differentiation from common marmoset (Callithrix jacchus) embryonic stem cells

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here, we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes, including tal1/scl, gata1, gata2, hoxB4, and lhx2, into CM ESCs. By immunochemical and morphological analyses, we demonstrated that overexpression of tal1/scl, but not the remaining genes, dramatically increased hematopoiesis of CM ESCs, resulting in multiple blood-cell lineages. Furthermore, flow cytometric analysis demonstrated that CD34, a hematopoietic stem/progenitor cell marker, was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells.     

A novel stepwise differentiation of functional pancreatic exocrine cells from embryonic stem cells

Cultivation of functional pancreatic cells isolated from adult mammalian pancreas remains difficult. We developed a differentiation protocol that gradually induced the formation of mouse pancreatic exocrine cells from embryonic stem cells (ESCs). This process mimicked in vivo pancreatic development by directing cells through definitive endoderm (DE), gut tube endoderm, and pancreatic progenitor cells to differentiated cells that expressed pancreatic exocrine enzymes. Mouse ESCs were cultured in hanging drops to form embryoid bodies. Treatment of embryoid bodies with activin A induced the formation of DE cells that expressed marker mRNAs Goosecoid and Mixl1 and that were double-positive with Foxa2 and Sox17 proteins. Subsequent treatment of the DE cells by retinoic acid induced the formation of gut tube endoderm cells that expressed the specific marker Hnf1b. Expression of Goosecoid and Mixl1 was downregulated during this period. Fibroblast growth factor 7 (FGF7) promoted differentiation of PDX1-expressing pancreatic progenitor cells that also expressed Foxa2 mRNA, an endodermal marker, suggesting derivation from the DE cells. Exocrine cell differentiation was induced with FGF7, glucagon-like peptide-1, and nicotinamide. The differentiated cells expressed mature pancreatic exocrine cell mRNAs, such as Amylase, Elastase, and Carboxypeptidase A. Additionally, they produced pancreatic elastase, amylase, carboxypeptidase A, and chymotrypsin proteins that were identified in cytoplasmic granules by immunocytochemistry. Active amylase was released into the medium. Moreover, FGF7 was associated with differentiation of pancreatic exocrine cells. The findings reported here offer a novel and effective process to develop pancreatic exocrine cells from ESCs.     

Ex vivo analysis of acinar and endocrine cell development in the human embryonic pancreas.

In contrast to the considerable body of data on pancreas development in rodents, information on pancreas development in humans is scant. We previously described a model in which mature beta cells developed from human embryonic pancreas: human embryonic pancreas was grafted under the kidney capsule of scid mice, beta cells were then seen to develop in the graft. Here, we showed that not only beta cells, but also other endocrine cells, acinar cells and ducts develop in this model. We then used this model to probe the mechanisms underlying acinar and beta cell development in the human embryonic pancreas. BrdU pulse/chase experiments produced evidence of clonal acinar cell development: the first acinar cells to appear proliferated, thereby expanding the acinar cell population. In contrast, beta cell development was regulated by the proliferation of pancreatic progenitor cells, followed by beta-cell differentiation. We then showed that early progenitors expressing PDX1 proliferated, whereas late endocrine progenitors expressing Ngn3 did not. This proliferative capacity of early endocrine progenitor cells in embryonic human pancreas may hold promise for obtaining human beta-cell expansion.     

Human Embryonic Stem Cell-extracts Inhibit the Differentiation and Function of Monocyte-derived Dendritic Cells

Embryonic stem cells (ESC) possess inherent properties of immune privilege with the capacity to evade allogeneic immune responses. Moreover, ESCs have been shown to prevent immune activation in response to third party antigen presenting cells in vitro and have the capacity to promote allograft survival in vivo. However, clinical use of human ESCs to treat immunological disorders may risk teratoma or ectopic tissue formation. Here, we show that cellular extracts from both human and mouse ESCs retain the immune modulatory properties of intact cells. ESC-extracts that contained 12–24 μg of total protein effectively prevented T cell proliferation in allogeneic mixed lymphocyte reactions (MLR), whereas control fibroblast extracts did not affect proliferation. Cellular mechanisms underlying hESC extract-mediated immune modulation involve the maturation of monocyte derived dendritic cells (mDC). hESC extract-treated mDCs had reduced surface expression of co-stimulatory and maturation markers CD80, HLA-DR and CD83 and secreted lower levels of IL12p40. Accordingly, hESC extract-treated DCs were found to be poor stimulators of purified allogeneic T cells compared to those DCs treated with vehicle or fibroblast extracts. Our results demonstrate that ESC extracts retain the immune modulatory properties of ESCs and for the first time demonstrates that ESC derived factors can inhibit human mDC maturation and function.     

SDF-1/CXCL12 enhances survival and chemotaxis of murine embryonic stem cells and production of primitive and definitive hematopoietic progenitor cells

Understanding embryonic stem cell (ESC) regulation is important for realizing how best to control their growth and differentiation ex vivo for potential therapeutic benefit. Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor, CXCR4, have been implicated as important regulators of a number of fetal and adult cell functions, including survival/antiapoptosis and migration/homing of hematopoietic stem and progenitor cells. We hypothesized that the SDF-1/CXCL12-CXCR4 axis would also be important for regulation of murine ESC functions. ESCs secreted low levels of SDF-1/CXCL12 and expressed low levels of CXCR4; however, both increased with differentiation of ESCs. Endogenously produced/released SDF-1/CXCL12 enhanced survival/antiapoptosis of ESCs in the presence of leukemia inhibitory factor but absence of serum, and survival/antiapoptosis was further enhanced by exogenous administration of SDF-1/CXCL12. Furthermore, SDF-1/CXCL12 induced chemotaxis of ESCs, and chemotaxis could be enhanced by diprotin A inhibition of CD26/dipeptidylpeptidase IV. Endogenous and exogenous SDF-1/CXCL12 enhanced embryoid body production of primitive and definitive erythroid, granulocyte-macrophage, and multipotential progenitors. SDF-1/CXCL12 did not noticeably affect production of hemangioblasts. These results demonstrate functional activities of SDF-1/CXCL12 on survival, chemotaxis, and hematopoietic differentiation of murine ESCs that may be relevant for their ex vivo manipulation.     

Brief report: efficient generation of hematopoietic precursors and progenitors from human pluripotent stem cell lines

By mimicking embryonic development of the hematopoietic system, we have developed an optimized in vitro differentiation protocol for the generation of precursors of hematopoietic lineages and primitive hematopoietic cells from human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs). Factors such as cytokines, extra cellular matrix components, and small molecules as well as the temporal association and concentration of these factors were tested on seven different human ESC and iPSC lines. We report the differentiation of up to 84% human CD45+ cells (average 41% ± 16%, from seven pluripotent lines) from the differentiation culture, including significant numbers of primitive CD45+/CD34+ and CD45+/CD34+/CD38- hematopoietic progenitors. Moreover, the numbers of hematopoietic progenitor cells generated, as measured by colony forming unit assays, were comparable to numbers obtained from fresh umbilical cord blood mononuclear cell isolates on a per CD45+ cell basis. Our approach demonstrates highly efficient generation of multipotent hematopoietic progenitors with among the highest efficiencies reported to date (CD45+/CD34+) using a single standardized differentiation protocol on several human ESC and iPSC lines. Our data add to the cumulating evidence for the existence of an in vitro derived precursor to the hematopoietic stem cell (HSC) with limited engrafting ability in transplanted mice but with multipotent hematopoietic potential. Because this protocol efficiently expands the preblood precursors and hematopoietic progenitors, it is ideal for testing novel factors for the generation and expansion of definitive HSCs with long-term repopulating ability.     

CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45-, Ter119-) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes.     

Human mast cells express receptors for IL-3, IL-5 and GM-CSF; a partial map of receptors on human mast cells cultured in vitro

BACKGROUND: Mast cells have long been recognized as the principal cell type that initiates the inflammatory response characteristic of acute allergic type 1 reactions. Our goal has been to further characterize maturation of progenitors to mast cells. METHODS: Mast cells were cultured from human cord blood derived CD133(+) progenitors. Mast cell function was tested using histamine release. During differentiation mast cells surface marker expression was monitored by flow cytometry. RESULTS: CD133(+) progenitors expressed the early haematopoietic and myeloid lineage markers CD34, CD117, CD13 and CD33. Mature mast cells expressed CD117, CD13 and CD33, and expression of the high affinity immunoglobulin E receptor FcepsilonRI increased during culture. Cytokine receptors interleukin (IL)-5R, IL-3R, granulocyte-macrophage-colony stimulating factor (GM-CSF)R and IL-18R were expressed at high levels during maturation. Chemokine receptors CXCR4 and CXCR2 were highly expressed on both newly purified CD133(+) cells and mature cells. CONCLUSION: Human mast cells can be cultured from a CD34(+)/CD117(+)/CD13(+)/CD33(+) progenitor cell population in cord blood that is tryptase and chymase negative. Developing and mature mast cells express a wide range of chemokine and cytokine receptors. We found high levels of expression of CD123, IL-5R and GM-CSF receptors, also found on eosinophils and basophils, and high levels of expression of the receptor for the inflammatory cytokine IL-18.     

Expression of CD26 (Dipeptidyl Peptidase IV) on Memory and Naive T Lymphocytes

It has been suggested that human CD26-positive T lymphocytes represent the memory pool of the cellular immune system. For proof of this suggestion we analysed the responsiveness of CD26-positive and CD26-negative T lymphocytes after antigenic stimulation in limiting dilution experiments. After stimulation with tetanus toxoid (TT) the number of proliferating cells within the CD26-positive subset was two- to sixfold higher than that within the CD26-negative subset. These differences in responsiveness were also detectable between CD4+/CD26+ and CD4+/CD26- T cells. To further investigate the memory character of the cells, human peripheral blood mononuclear cells were stimulated with TT for 7 or 14 days. Thereafter, CD26+ and CD26- T cells were isolated and restimulated with TT in limiting dilution experiments. Responding cells were found not only within the CD26-positive subset but also within the CD26-negative subset, and their number increased with time. Surface marker analysis of freshly isolated human T lymphocytes or T-cell clones indicated that CD26 is not a stable cell surface marker. Furthermore, CD26 is both absent and present on CD29-positive or CD45RA-positive cells, indicating no association of CD26 with surface markers for memory or naive T cells, respectively. These results strongly argue against the hypothesis that CD26-positive T cells represent the memory pool. We conclude that CD26 is an activation marker of T lymphocytes, which is associated with reactivity on naive and memory cells.     

Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro

Embryonic stem cells (ESCs) have the potential to serve as a renewable source of transplantable tissue-specific stem cells. However, the molecular cues necessary to direct the differentiation of ESCs toward specific cell lineages remain obscure. Here we report the successful induction of ESC differentiation into mature functional T lymphocytes with a simple in vitro coculture system. The directed differentiation of ESCs into T cells required the engagement of Notch receptors by Delta-like 1 ligand (DL1) expressed on the OP9-DL1 stromal cell line. We found a normal program of T cell differentiation in ESC-OP9-DL1 cell cocultures. ESC-derived T cell progenitors effectively reconstituted the T cell compartment of immunodeficient mice, enabling an effective response to a viral infection. These findings provide a powerful tool for the molecular analysis of T cell development and open new avenues for the development of immunotherapeutic approaches using defined sources of stem cells.