Characterization of human embryonic stem cell-derived hematopoietic progenitor phenotype

Differentiation of human embryonic stem cells (hESCs) into hematopoietic lineages using various methods has been reported. However, the phenotype that precisely defines the hematopoietic progenitor compartment with clonogenic activities has yet to be determined. Here, we measured and characterized progenitor function of subfractions of cells prospectively isolated from human embryoid bodies (hEBs) during hematopoietic differentiation basing on surface markers CD45, CD34, CD43, and CD38. We report that hematopoietic progenitors predominantly resided in the CD45⁺ subset. CD43⁺ cells lacking CD45 expression were largely devoid of progenitor activity. However, progenitor activity and multipotentiality was more enriched in CD45⁺ cells co-expressing CD43. CD45⁺ subset co-expressing CD34 but lacking CD38 expression (CD45⁺CD34⁺CD38^-) were further enriched for CFU capacity compared to the CD45⁺CD34⁺CD38⁺ subset. Our study demonstrates a role of CD43 in enriching hematopoietic progenitors derived from hEBs and reveals a hierarchical organization of hESC-derived hematopoietic progenitor compartments defined by phenotypic markers.     

Endothelial origin of mesenchymal stem cells

Mesenchymal stem/stromal cells (MSCs) are fibroblastoid cells capable of long-term expansion and skeletogenic differentiation. While MSCs are known to originate from neural crest and mesoderm, immediate mesodermal precursors that give rise to MSCs have not been characterized. Recently, using human embryonic stem cells (hESCs), we demonstrated that mesodermal MSCs arise from APLNR⁺ precursors with angiogenic potential, mesenchymoangioblasts, which can be identified by FGF2-dependent colony-forming assay in serum-free semisolid medium. In this overview we provide additional insights on cellular pathways leading to MSC establishment from mesoderm, with special emphasis on endothelial-mesenchymal transition as a critical step in MSC formation. In addition, we highlight an essential role of FGF2 in induction of angiogenic cells with potential to transform into MSCs (mesenchymoangioblasts) or hematopoietic cells (hemangioblasts) from mesoderm, and discuss correlations of our in vitro findings with the course of angioblast development during embryogenesis.Key words: mesenchymoangioblast, hemangioblast, human embryonic stem cells, endothelial-mesenchymal transition, epithelial-mesenchymal transition, mesenchymal stem cells, endothelial cells, apelin receptor, FGFMesenchymal stem/stromal cells (MSCs) are defined as multipotent fibroblastoid cells that give rise to cells of the skeletal connective tissue including osteoblasts, chondrocytes and adipocytes.^1–4 Although MSCs were described more than 40 years ago and are widely used for cellular therapies, very little knowledge exists regarding the developmental origins of MSCs in the embryo, the hierarchy of MSC progenitors or heterogeneity of MSCs within tissues. It has been demonstrated that during embryonic development, MSCs arise from a two major sources: neural crest and mesoderm.^5–7 Using Cre-recombinase lineage tracing experiments, Takashima et al. identified Sox1⁺ neuroepithelium as pre-cursors of MSCs of neural crest origin. However, direct precursors of mesoderm-derived MSCs were unknown. To identify these precursors, we employed human embryonic stem cells (hESCs) directed toward mesendodermal differentiation in coculture with mouse bone marrow stromal cells OP9,⁸ using the experimental approach depicted in Figure 1. As shown in this differentiation system, mesoderm reminiscent of lateral plate/extraembryonic mesoderm in the embryo can be identified by expression of apelin receptor (APLNR), otherwise known as angiotensin receptor like-1 receptor. Because we observed a positive selective effect of FGF2 on production of mesenchymal cells from hESCs in OP9 coculture, we decided to test whether FGF2 can induce the formation of colonies with mesenchymal potential from APLNR⁺ mesodermal cells. Indeed, when we isolated APLNR⁺ cells from hESCs differentiated on OP9 for 2 days and placed them in serum-free semisolid medium containing FGF2, we observed the formation of sharply-circumscribed spheroid colonies formed by tightly packed cells with a gene expression profile representative of embryonic mesenchyme originating from lateral plate/extraembryonic mesoderm and CD140a⁺CD146⁺C D90⁺CD56⁺CD166⁺CD31⁻CD43⁻CD45⁻ phenotype typical of mesenchymal cells. Based on cellular composition, we designated these colonies as mesenchymal (MS) colonies and cells forming these colonies as MS colony-forming cells (MS-CFCs). MS colony formation required serum-free medium and was solely dependent on FGF2 as a colony-forming factor. MS colonies were significantly enhanced by PDGF-BB, but suppressed by VEGF, TGFβ1 and Activin A. When transferred to the adherent cultures in serum-free medium with FGF2, individual MS colonies gave rise to multi-potential mesenchymal cell lines with typical phenotype (CD146⁺ CD105⁺ CD73⁺ CD31⁻ CD43/45⁻), differentiation (chondro-, osteo- and adipogenesis) and robust proliferation (>80 doublings) potentials. Using single cell deposition assay, chimeric hESC lines and time-lapse studies we demonstrated the clonality/single cell origin of MS colonies.Open in a separate windowFigure 1Schematic diagram of the experimental approach used to identify precursors and cellular events leading to formation of mesoderm-derived MSCs. hESCs were committed to mesendodermal differentiation through coculture with OP9 for 2 days. APLNR⁺ mesodermal cells were selected using magnetic sorting. In serum-free semisolid medium, APLNR⁺ cells grew into FGF2-dependent compact spheroid colonies composed of mesenchymal cells. MS colonies were formed through establishment of tightly-packed single cell-derived cores (day 3 of clonogenic culture), which expanded into spheroid colonies (days 6 and 12 of clonogenic culture). To evaluate differentiation potential, MS colonies were collected at different stages of clonogenic culture and placed on OP9. The presence of endothelial and mesenchymal cells after coculture of MS colonies with OP9 was evaluated by flow cytometry and immunofluorescence. In addition, colonies at core stage (day 3 of clonogenic culture) and mature colonies (day 12 of clonogenic cultures) were collected for molecular profiling studies. To generate clonal MSC lines, individual mature colonies were plated on the collagen/fibronectin-coated plastic and cultured in presence of FGF2.MS-CFCs could be detected only transiently, with a major peak on day 2 of hESC differentiation and disappeared after 4 days of differentiation. Notably, MS-CFC activity was developed prior to the expression of CD73 and CD105 MSC markers and upregulation of MSC-related genes, i.e., before onset of mesenchymogenesis. APLNR⁺ cells isolated from hESC cultures differentiated for 3 days also formed colonies in response to FGF2; however, the vast majority of these colonies were composed of blood cells and had a morphology similar to the previously described blast (BL) or hemangioblast colonies, which identify a common precursor for hematopoietic and endothelial cells.^9,10To fully evaluate the differentiation potential of MS colonies, we collected these colonies from semisolid cultures and placed them back on OP9 feeders, which are known to support development of a broad range of mesodermal lineage cells, including hematopoietic, vascular and cardiac.^11–13 Using this approach, we confirmed that individual BL colonies possess hemangioblastic potential, i.e., generate both hematopoietic and endothelial cells. When MS colonies were picked from clonogenic cultures and cultured on OP9, we found that the majority of cells differentiated into CD146⁺CD31⁻CD43/CD45⁻ mesenchymal cells as expected. However, we also discovered that MS colonies gave rise to CD31/VE-cadherin⁺CD43/45⁻ endothelial cells, indicating that MS colonies similar to BL colonies possess endothelial potential. The endothelial potential of MS colonies was also confirmed by demonstration of tube formation by MS colonies grown on Matrigel. In contrast, MSC lines derived from MS colonies did not produce any endothelial cells after coculture with OP9 indicating a progressive restriction of differentiation potential following MSC formation. Because single MS-CFC shows potential to form endothelium and MSCs, we designated the MSC precursor identified by this colony-forming assay as mesenchymoangioblast.To define more precisely the cellular events leading to establishing MSCs, we examined the formation of MS colonies using time-lapse cinematography and analyzed the kinetic of their angiogenic potential. As demonstrated by time-lapse studies, APLNR⁺ mesodermal cells placed in semisolid medium possessed a high motility, which was more pronounced before and during the first cell division. Following several divisions, single APLNR⁺ cells formed a core, an immotile structure composed of a small number of tightly packed cells. While APLNR⁺ mesodermal cells lacked endothelial gene expression, molecular profiling of MS colonies at the core stage revealed that these cells acquired angioblastic gene expression profile as indicated by upregulation of FLT1, TEK, CDH5 (VE-cadherin), PECAM1 (CD31), FLI1, SELE (ELAM-1) and ICAM2 endothelial genes. When we collected MS cores (day 3 of clonogenic culture) and placed them on OP9, they formed predominantly VE-cadherin⁺ endothelial clusters, strongly indicating the endothelial nature of the core-forming cells. Subsequently, cells at the periphery of the core underwent endothelial-mesenchymal transition (EndMT) and formed a shell of tightly packed spindle-like cells around the core. When we picked colonies at this stage (day 6 of colony-forming culture) and placed them on OP9, most of the colonies (>70%) grew cell clusters composed of endothelial and mesenchymal cells. In contrast, mature MS colonies collected on day 12 of clonogenic culture formed predominantly clusters of mesenchymal cells, indicating a progressive loss of endothelial potential following colony maturation. Although no CD31 expression was detected in the mesenchymal cells composing mature MS colonies, these cells retained several endothelial traits including surface expression of endothelial tyrosine kinase (TEK or TIE2), FLT1 (VEGFR1) and endomucin. The critical role of EndMT in MS colony formation and MSC development was also congruous with our observation of the suppressive effect of VEGF, a known inhibitor of EndMT,^14,15 on MS colonies. When VEGF was added to MS clonogenic cultures, hESC-derived mesodermal cells were capable of forming angiogenic cores; however, these cores did not transform into mesenchymal cells, indicating that VEGF abrogates MS colony development at the core stage through inhibition of EndoMT. The schematic diagram demonstrating development of mesodermal MSCs is presented in Figure 2.Open in a separate windowFigure 2A model of mesoderm-derived MSC development from hESCs. Coculture with OP9 stromal cells predominantly induces hESC differentiation toward APLNR⁺ mesoderm. APLNR⁺ population contains angiogenic mesodermal precursors with either mesenchymal (mesenchymoangioblast) or hematopoietic (hemangioblast) potentials. Mesenchymoangioblasts and hemangioblasts arise sequentially during differentiation and can be revealed by MS and BL colony formation in response to FGF2. Development of MS and BL colonies in semisolid media proceed through a core stage at which APLNR⁺ cells form clusters of tightly packed cells with angiogenic potential. Subsequently, core-forming cells undergo EndMT giving rise to mesenchymal cells, which form a shell around the core developing into a mature MS colony. VEGF, EndMT inhibitor, blocks MS colony-formation at core stage. The ability of MS-CFCs to generate mesenchymal and endothelial cells can be revealed by coculture of individual colonies with OP9. Similar to MS colonies, BL colonies are formed through establishment of angiogenic core. However, hemangioblast core-forming cells undergo endothelial-hematopoietic transition and grew hematopoietic cells around the core.The close relationship between endothelial and hematopoietic cell development was recognized more than 130 years ago (reviewed by ref. ¹⁶) and confirmed in multiple modern studies.^9,17–22 However, the association between endothelial pre-cursors and MSCs during development was not well established, although cells with endothelial and mural cell potential were identified²³ and the critical role of EndMT in the formation of endocardial cushion²⁴ and testicular cords²⁵ in the embryo was acknowledged. Our hESC-based in vitro studies indicated that formation of mesodermal MSCs proceed through the endothelial stage and likely included at least two successive cycles of cell transitions. Initially APLNR⁺ mesoderm, which consists of fibroblast-like migratory cells, give rise to core structures composed of tightly packed endothelial cells in response to FGF2. Subsequently, endothelial cells forming cores undergo epithelial-mesenchymal transition, i.e., EndMT and form MSCs. The question remains how well this in vitro model reflects in vivo development. Although only sparse data exist regarding MSC precursors in the embryo, development of angiogenic hematopoietic precursors, hemangioblasts was studied more extensively in mammals and birds, and therefore parallels between in vivo and in vitro studies can be drawn. As we demonstrated,⁸ APLNR⁺ mesodermal cells collected from hESCs differentiated on OP9 for 3 days formed disperse BL colonies that identify hemangioblasts in vivo and in vitro.^9,26 Similar to MS colonies, the development of BL colonies required FGF2 and proceeded through angiogenic core formation. However, in contrast to MS cores, BL cores transformed into blood cells, i.e., underwent endothelial-hematopoietic transformation (see Fig. 2). Importantly, in vivo studies identified FGF2 as the essential factor in hemangioblast induction²⁷ analogous to our in vitro observation. In chicken embryo, the activation of FGF signaling leads to aggregation of migrating mesodermal cells adjacent to the endoderm, upregulation of VEGFR2 (KDR) expression, and subsequent formation of angioblasts and hemangioblasts.^28–30 This sequence of events leading to hemangioblast development in vivo considerably resembles what we observed in vitro, and highly suggests accurate recapitulation of embryonic development by our hESC differentiation model. Therefore, searching for an in vivo equivalent of mesenchymonagioblast would be a reasonable next step.In addition to embryonic development, EndMT is also implicated in several pathologies including cancer progression and development of cardiac and renal fibrosis.^31–34 Recently, Olsen group revealed that endothelial cells could be transformed directly into MSCs through overexpression of ALK2 or its activation by TGFβ2 or BMP4,¹⁵ indicating that endothelial cells could be an important source of MSCs in postnatal life. Conversely, the transition from MSCs to endothelial cells, has been also described in reference ³⁵. Based on these observations, a cycle of cell-fate transition from endothelium to MSCs and back to endothelium was proposed as a circuit controlling stem cell state.³⁶ Since multiple parallels could be drawn between EndMT described in adult tissues and during hESC differentiation, one may wonder whether bipotential cells with endothelial and MSC potential similar to embryonic mesenchymoangioblasts are present and constitute an important element of EndMT circuit in adults.In conclusion, the identification of mesenchymoangioblast as a clonogenic precursor of mesoderm-derived MSCs is an important step toward defining pathways of MSC development and specification. In addition, the demonstration of MSC formation from mesoderm through EndMT provides new insights into the mechanisms involved in establishment of MSCs.     

In vitro T lymphopoiesis: A model system for stem cell gene therapy for AIDS

Abstract: Stable introduction of therapeutic genes into hematopoietic stem cells has the potential to reconstitute immunity in individuals with HIV infection. However, many important questions regarding the safety and efficacy of this approach remain unanswered and may be addressed in a non-human primate model. To facilitate evaluation of expression of foreign genes in T cells derived from transduced hematopoietic progenitor cells, we have established a culture system that supports the differentiation of rhesus macaque and human CD34⁺ bone marrow derived cells into mature T cells. Thymic stromal monolayers were prepared from the adherent cell fraction of collagenase digested fetal or neonatal thymus. After 10–14 days, purified rhesus CD34⁺ bone marrow-derived cells cultured on thymic stromal monolayers yielded CD3⁺CD4⁺CD8⁺, CD3⁺CD4⁺CD8^?, and CD3⁺CD4^?CD8⁺ cells. Following stimulation with mitogens, these T cells derived from CD34⁺ cells could be expanded over 1,000-fold and maintained in culture for up to 20 weeks. We next evaluated the ability of rhesus CD34⁺ cells transduced with a retroviral vector containing the marker gene neo to undergo in vitro T cell differentiation. CD34⁺ cells transduced in the presence of bone marrow stroma and then cultured on rhesus thymic stroma resulted in T cells containing the retroviral marker gene. These studies should facilitate both in vitro and in vivo studies of hematopoietic stem cell therapeutic strategies for AIDS.     

The IL-2A receptor pathway and its role in lymphocyte differentiation and function

Murine myeloid cells are developed from hemopoietic stem/progenitor cells. Different types of progenitor cells have variable differentiation potentials. Among the ten main types of cells differentiated from lymphoid progenitor cells, regulatory T cells (Tregs), an important cell subpopulation regulating immune and inflammatory responses, arise from the hematopoietic stem cells in the bone marrow. Tregs then differentiate into T lymphocytes and migrate to the thymus and finally generate Treg subsets, which are subsequently activated and regulated by inflammatory cytokines in the peripheral blood. Tregs also have different phenotypes and immunomodulatory functions. The cytokine interleukin-2/interleukin-2 receptor (IL-2/IL-2R) pathway is an important regulatory signaling pathway of Tregs. Besides, different types of CD4⁺ and CD8⁺ cells have different immune effects in the absence of IL-2. IL-2R consists of three subunits, α chain (CD25), β chain (CD122), and γ chain (CD132). Different subunit combinations have different effects on the activation of immune cells. Multiple studies have shown that IL2RA deficiency has various effects on the immune function in mice. This article reviews the subunit composition and signaling pathway of IL-2R, the classification of Tregs in a murine myeloid cell line and the regulatory effect of IL-2/IL-2R on them, the regulatory impact and signaling mechanism of IL-2/IL-2R on CD4⁺/CD8⁺ lymphocyte differentiation, the primary manifestations and molecular mechanism of immune dysfunction in IL-2- and IL-2R-deficient mice, soluble IL-2Rα as a biomarker for diagnosis, prognosis and therapeutic efficacy of treatment in immune system disorders, and the development and clinical application of IL-2 mutants.     

Characterization of developmental pathway of natural killer cells from embryonic stem cells in vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(-) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(-) EB cells showed that CD45(+)Mac-1(-)Ter119(-) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(-)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(-) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(-) and they rapidly acquire CD122 as they differentiate along the NK lineage.     

T-cell potential of human adult and cord blood hemopoietic stem cells expanded with the use of aryl hydrocarbon receptor antagonists

Background aimsExpansion of hemopoietic stem cells (HSCs) in vitro is a potential strategy for improving transplant outcomes, but expansion methods tend to promote differentiation and loss of stem cell potential. Aryl hydrocarbon receptor antagonists (AhRAs) have recently been shown to protect HSC stemness during expansion; however, little is known of the T-cell regenerative capacity of AhRA-expanded HSCs. In this study, we confirm the protective effect of two commercially available AhRA compounds on HSCs from both cord blood (CB) and adult samples and assess the T-lymphocyte potential of the expanded cells.MethodsAdult mobilized peripheral blood and CB samples were purified to CD34⁺ cells, which were expanded in vitro with cytokines and AhRAs. After 14 d, CD34⁺ cells were re-isolated and then grown on in OP9Delta co-culture under conditions that allow T-lymphocyte differentiation. Cells were monitored weekly for T-lineage markers by flow cytometry.ResultsBoth AhRA compounds promoted maintenance of CD34 expression during 2 weeks of proliferation with growth factors, although adult cells proliferated markedly less than CB cells. AhRA-expanded CD34⁺ cells from CB differentiated to T cells on OP9Delta co-culture with the same rate and time course as untreated cells. Adult cells, by contrast, had reduced differentiation to T cells, with donor-dependent variable responses.ConclusionsThis study shows that whereas AhRA treatment is effective in CB samples, expansion of adult HSCs is less successful and reflects their inherent poor potential in T-cell generation.     

Generation of Hematopoietic Stem Cells from Purified Embryonic Endothelial Cells by a Simple and Efficient Strategy

Recent progress by versatile approaches supports the new hypothesis that multi-potent hematopoietic stein cells （HSCs） are directly formed from a rare population of endothelial cells in mid-gestation mouse embryos. This process is therefore known as the endothelial-to- hematopoietic transition （EHT）. Nevertheless, there is no functional evidence that documents the HSC transition from purified endothelial cells. In this study, we developed an OP9-DLl-based co-culture system that was able to facilitate the HSC specification and/or expansion in vitro of mouse embryonic day 10.5 （El0.5） Tie2~ cells remarkably. Then, the immunophenotypically defined endothelial ceils were harvested by a combination of surface markers （Flkl＋CD31 ~CD41 CD45 Ter119 ） from the caudal half of EI0.0-EI 1.0 mouse embryos. The transplantation of the endothelia/OP9-DL1 co-cultures led to long-term, high-level, multi-lineage, and multi-organ he- matopoietic reconstitution in the irradiated adult recipients. The induced HSC activity was initially observed at El0.5, and a significant increase was detected at El 1.0, which suggests a temporally specific regulation. Taken together, tbr the first time, we provide functional evidence showing the HSC potential of purified embryonic endothelial cells, which is indispensable for the emerging EHT concept. Moreover, the newly defined co-culture system will aid the exploration of the key molecules governing the HSC transition from embryonic and even postnatal endothelial cells, which has enormous significance in basic and translational research.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Role of Reactive Oxygen Species in the Radiation Response of Human Hematopoietic Stem/Progenitor Cells

Hematopoietic stem/progenitor cells (HSPCs), which are present in small numbers in hematopoietic tissues, can differentiate into all hematopoietic lineages and self-renew to maintain their undifferentiated phenotype. HSPCs are extremely sensitive to oxidative stressors such as anti-cancer agents, radiation, and the extensive accumulation of reactive oxygen species (ROS). The quiescence and stemness of HSPCs are maintained by the regulation of mitochondrial biogenesis, ROS, and energy homeostasis in a special microenvironment called the stem cell niche. The present study evaluated the relationship between the production of intracellular ROS and mitochondrial function during the proliferation and differentiation of X-irradiated CD34⁺ cells prepared from human placental/umbilical cord blood HSPCs. Highly purified CD34⁺ HSPCs exposed to X-rays were cultured in liquid and semi-solid medium supplemented with hematopoietic cytokines. X-irradiated CD34⁺ HSPCs treated with hematopoietic cytokines, which promote their proliferation and differentiation, exhibited dramatically suppressed cell growth and clonogenic potential. The amount of intracellular ROS in X-irradiated CD34⁺ HSPCs was significantly higher than that in non-irradiated cells during the culture period. However, neither the intracellular mitochondrial content nor the mitochondrial superoxide production was elevated in X-irradiated CD34⁺ HSPCs compared with non-irradiated cells. Radiation-induced gamma-H2AX expression was observed immediately following exposure to 4 Gy of X-rays and gradually decreased during the culture period. This study reveals that X-irradiation can increase persistent intracellular ROS in human CD34⁺ HSPCs, which may not result from mitochondrial ROS due to mitochondrial dysfunction, and indicates that substantial DNA double-strand breakage can critically reduce the stem cell function.     

Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells

In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin(-)CD34(+)CD43(+)CD45(+) multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5-19 d of culture with cytokines, depending on the cell type.     

Stimulation of wnt/ß-catenin pathway in human CD8(+) T lymphocytes from blood and lung tumors leads to a shared young/memory phenotype

Cancer can be treated by adoptive cell transfer (ACT) of T lymphocytes. However, how to optimally raise human T cells to a differentiation state allowing the best persistence in ACT is a challenge. It is possible to differentiate mouse CD8⁺ T cells towards stem cell-like memory (T_SCM) phenotype upon TCR stimulation with Wnt/ß-catenin pathway activation. Here, we evaluated if T_SCM can be obtained from human mature CD8⁺ T cells following TCR and Wnt/ß-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119), which inhibits the glycogen synthase kinase-3β (GSK-3β), key inhibitor of the Wnt pathway. Human CD8⁺ T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL), and treated with TWS119 gave rise to CD62L⁺CD45RA⁺ cells, indicative of early differentiated stage, also expressing CD127 which is normally found on memory cells, and CD133, an hematopoietic stem cell marker. T_SCM cells raised from either TIL or blood secreted numerous inflammatory mediators, but in lower amounts than those measured without TWS119. Finally, generated T_SCM CD8⁺ T cells expressed elevated Bcl-2 and no detectable caspase-3 activity, suggesting increased persistence. Our data support a role for Wnt/ß-catenin pathway in promoting the T_SCM subset in human CD8⁺ T cells from TIL and the periphery, which are relevant for ACT.     

Phosphodiesterase 4A confers resistance to PGE2‐mediated suppression in CD25+/CD54+ NK cells

Inadequate persistence of tumor‐infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL‐2 or IL‐15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL‐15, in contrast to IL‐2, enriches for CD25⁺/CD54⁺ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25⁺/CD54⁺ NK cells for adoptive cell therapy should be considered.     

Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2

Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30^Hi-GCTM-2^Hi hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30^Neg-GCTM-2^Neg). In a further study, pluripotent stem cell-free samples of differentiated TG30^Neg-GCTM-2^Neg cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30^Hi-GCTM-2^Hi hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.