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Endothelial-to-hematopoietic transition (EHT) is a unique morphogenic event in which flat, adherent hemogenic endothelial (HE) cells acquire round, non-adherent blood cell morphology. Investigating the mechanisms of EHT is critical for understanding the development of hematopoietic stem cells (HSCs) and the entirety of the adult immune system, and advancing technologies for manufacturing blood cells from human pluripotent stem cells (hPSCs). Here we describe a protocol to (a) generate and isolate subsets of HE from hPSCs, (b) assess EHT and hematopoietic potential of HE subsets in bulk cultures and at the single-cell level, and (c) evaluate the role of NOTCH signaling during HE specification and EHT. The generation of HE from hPSCs and EHT bulk cultures are performed in xenogen- and feeder-free system, providing the unique advantage of being able to investigate the role of individual signaling factors during EHT and the definitive lympho-myeloid cell specification from hPSCs.

Summary Human induced pluripotent stem cells (hiPSCs) can serve as a versatile and scalable source of neutrophils for biomedical research and transfusion therapies. Here we describe a rapid efficient serum- and xenogen-free protocol for neutrophil generation, which is based on direct hematoendothelial programming of hiPSCs using ETV2-modified mRNA. Culture of ETV2-induced hematoendothelial progenitors in the presence of GM-CSF, FGF2, and UM171 led to continuous production of generous amounts of CD34+CD33+ myeloid progenitors which could be harvested every 8–10 days for up to 30 days of culture. Subsequently, myeloid progenitors were differentiated into neutrophils in the presence of G-CSF and the retinoic acid agonist Am580. Neutrophils obtained in these conditions displayed a typical somatic neutrophil morphology, produced reactive oxygen species, formed neutrophil extracellular traps and possessed phagocytic and chemotactic activities. Overall, this technology offers an opportunity to generate a significant number of neutrophils as soon as 14 days after initiation of differentiation., Graphical Abstract, Highlights • ETV2 mmRNA directly programs hPSCs into hemogenic endothelium (HE) • ETV2-induced HE possesses robust myeloid potential • ETV2 mmRNA rapid neutrophil differentiation protocol in defined conditions is provided • ETV2 mmRNA-induced neutrophils are functionally similar to in-vivo-derived cells, In this article, Slukvin and colleagues describes a protocol for a rapid efficient feeder-, serum-, and xenogen-free protocol for neutrophil generation from hiPSCs, which is based on direct hematoendothelial programming of hiPSCs using ETV2-modified mRNA.

SUMMARY This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using ETV2 modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function. For complete details on the use and execution of this protocol, please refer to Brok-Volchanskaya et al. (2019)., Graphical Abstract This protocol describes a rapid and efficient feeder-, serum-, and xeno-free method for neutrophil generation from hiPSCs using ETV2 modified mRNA (mmRNA), which directs hematoendothelial programming of hiPSCs. Hematoendothelial progenitors were cultured with GM-CSF, FGF-2, and UM171 to expand myelomonocytic progenitors, followed by treatment with G-CSF and retinoic acid agonist Am580 to induce neutrophil maturation. This protocol is suitable for generating functional neutrophils from iPSCs to interrogate the role of genes in a neutrophil development and function.

Summary The transcriptional factor GATA2 is required for blood and hematopoietic stem cell formation during the hemogenic endothelium (HE) stage of development in the embryo. However, it is unclear if GATA2 controls HE lineage specification or if it solely regulates endothelial-to-hematopoietic transition (EHT). To address this problem, we innovated a unique system, which involved generating GATA2 knockout human embryonic stem cell (hESC) lines with conditional GATA2 expression (iG2−/− hESCs). We demonstrated that GATA2 activity is not required for VE-cadherin+CD43−CD73+ non-HE or VE-cadherin+CD43−CD73– HE generation and subsequent HE diversification into DLL4+ arterial and DLL4– non-arterial lineages. However, GATA2 is primarily needed for HE to undergo EHT. Forced expression of GATA2 in non-HE failed to induce blood formation. The lack of GATA2 requirement for generation of HE and non-HE indicates the critical role of GATA2-independent pathways in specification of these two distinct endothelial lineages., Graphical Abstract, Highlights • GATA2 is not required for hemogenic endothelium (HE) specification • GATA2 regulates endothelial-to-hematopoietic transition in HE • GATA2 fails to enforce hematopoietic program in non-HE • Distinct GATA2-regulated networks are established in HE and non-HE, In this article, Slukvin and colleagues show that GATA2 transcription factor is dispensable for hemogenic endothelium (HE) specification and diversification. However, GATA2 is primarily needed for HE to undergo endothelial-to-hematopoietic transition. They also revealed differences in the GATA2-regulated network in HE and non-HE.

Specification of endothelial cells (ECs) into arterial, venous, and lymphatic cells is a crucial process of vascular development, and expanding our knowledge about EC specification from human pluripotent stem cells (hPSCs) will aid the design of optimal strategies for producing desired types of ECs for therapies. In our prior studies, we revealed that hPSC-derived VE-cadherin(V)(+)CD31(+)CD34(+) ECs are heterogeneous and include at least three major subsets with distinct hemogenic properties: V(+)CD43/235a(−)CD73(−) hemogenic endothelial progenitors (HEPs), V(+)CD43(lo)CD235a(+)73(−) angiogenic hematopoietic progenitors (AHPs), and V(+)CD43/235a(−)73(+) non-HEPs. In this study, using angiogenesis assays, we demonstrated that ECs within these subsets have distinct endothelial colony- and tube-forming properties, proliferative and migratory properties, and endothelial nitric oxide synthase and inflammatory cytokine production potentials. Culture of isolated subsets in arterial, venous, and lymphatic conditions revealed that AHPs are skewed toward lymphatic, HEPs toward arterial, and non-HEPs toward venous differentiation in vitro. These findings suggest that selection and enhancement of production of a particular EC subset may aid in generating desirable EC populations with arterial, venous, or lymphatic properties from hPSCs.

SUMMARY Elucidating the pathways that lead to vasculogenic cells, and being able to identify their progenitors and lineage-restricted cells, is critical to the establishment of human pluripotent stem cell (hPSC) models for vascular diseases and development of vascular therapies. Here, we find that mesoderm-derived pericytes (PCs) and smooth muscle cells (SMCs) originate from a clonal mesenchymal progenitor mesenchymoangioblast (MB). In clonogenic cultures, MBs differentiate into primitive PDGFRβ+ CD271+CD73− mesenchymal progenitors, which give rise to proliferative PCs, SMCs, and mesenchymal stem/stromal cells. MB-derived PCs can be further specified to CD274+ capillary and DLK1+ arteriolar PCs with a proinflammatory and contractile phenotype, respectively. SMC maturation was induced using a MEK inhibitor. Establishing the vasculogenic lineage tree, along with identification of stage- and lineage-specific markers, provides a platform for interrogating the molecular mechanisms that regulate vasculogenic cell specification and diversification and manufacturing well-defined mural cell populations for vascular engineering and cellular therapies from hPSCs., In Brief Kumar et al. find that mesodermal pericytes and smooth muscle cells in human pluripotent stem cell cultures originate from a common endothelial and mesenchymal cell precursor, the mesenchymoangioblast. They show how different lineages of mural cells are specified from mesenchymoangioblasts and define stage- and lineage-specific markers for vasculogenic cells., Graphical Abstract

Scaling up blood cell production from hPSCs is critical to advancing hPSC technologies for blood transfusion, immunotherapy, and transplantation. Here we explored the potential of the HSC agonist pyrimido-indole derivative UM171, to expand hematopoietic progenitors (HPs) derived from hPSCs in chemically defined conditions. We revealed that culture of hPSC-HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3L, IL3 and IL6) in the presence of UM171 predominantly expanded HPs with a unique CD34+CD41aloCD45+ phenotype that were enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4, in the presence of SCF, FLT3L, and IL7, UM171 selectively expanded CD34+CD45+CD7+ lymphoid progenitors with NK cell potential, and increased NK cell output up to 10-fold. These studies should improve our understanding of the effect of UM171 on de novo generated HPs, and facilitate development of protocols for robust granulocyte and lymphoid cell production from hPSCs, for adoptive immunotherapies.

SUMMARY SOX17 has been implicated in arterial specification and the maintenance of hematopoietic stem cells (HSCs) in the murine embryo. However, knowledge about molecular pathways and stage-specific effects of SOX17 in humans remains limited. Here, using SOX17-knockout and SOX17-inducible human pluripotent stem cells (hPSCs), paired with molecular profiling studies, we reveal that SOX17 is a master regulator of HOXA and arterial programs in hemogenic endothelium (HE) and is required for the specification of HE with robust lympho-myeloid potential and DLL4+CXCR4+ phenotype resembling arterial HE at the sites of HSC emergence. Along with the activation of NOTCH signaling, SOX17 directly activates CDX2 expression, leading to the upregulation of the HOXA cluster genes. Since deficiencies in HOXA and NOTCH signaling contribute to the impaired in vivo engraftment of hPSC-derived hematopoietic cells, the identification of SOX17 as a key regulator linking arterial and HOXA programs in HE may help to program HSC fate from hPSCs., Graphical Abstract, In Brief Jung et al. report that SOX17 is a critical upstream factor that is required for the activation and linkage of HOXA and arterial programs in the hemogenic endothelium and for establishing DLL4+CXCR4+ arterial hemogenic endothelium with definitive lympho-myeloid potential. These SOX17 effects are mediated through the activation of CDX2 and NOTCH signaling.

Human embryonic stem cell line WA0 1 was genetically modified using zinc-finger nucleases and the PiggyBac/transponson system to introduce a fluorescence reporter for VE-cadherin (VEC; tdTomato) and CD43 (eGFP). Phenotypic and functional assays for pluripotency revealed the modified hES cell reporter lines remained normal. When the cells were differentiated into hematoendothelial lineages, either by directed differentiation or direct reprogramming, flow cytometric and fluorescence microscopy showed that VEC + endothelial cells express tdTomato and CD43 + hematopoietic progenitors express eGFP.

Human pluripotent stem cells (hPSCs) have been suggested as a potential source for the de novo production of blood cells for transfusion, immunotherapies, and transplantation. However, even with advanced hematopoietic differentiation methods, the primitive and myeloid-restricted waves of hematopoiesis dominate in hPSC differentiation cultures, whereas cell surface markers to distinguish these waves of hematopoiesis from lympho-myeloid hematopoiesis remain unknown. In the embryo, hematopoietic stem cells (HSCs) arise from hemogenic endothelium (HE) lining arteries, but not veins. This observation led to a long-standing hypothesis that arterial specification is an essential prerequisite to initiate the HSC program. It has also been established that lymphoid potential in the yolk sac and extraembryonic vasculature is mostly confined to arteries, whereas myeloid-restricted hematopoiesis is not specific to arterial vessels. Here, we review how the link between arterialization and the subsequent definitive multilineage hematopoietic program can be exploited to identify HE enriched in lymphoid progenitors and aid in in vitro approaches to enhance the production of lymphoid cells and potentially HSCs from hPSCs. We also discuss alternative models of hematopoietic specification at arterial sites and recent advances in our understanding of hematopoietic development and the production of engraftable hematopoietic cells from hPSCs.

Identification of transcription factors that directly convert pluripotent stem cells (PSCs) into endothelial and blood cells and advances in the chemical modifications of messenger RNA (mRNA) offer alternative nucleic acid-based transgene-free approach for scalable production of these cells for drug screening and therapeutic purposes. Here we evaluated the effect of 5′ and 3′ RNA untranslated regions (UTRs) on translational efficiency of chemically-modified synthetic mRNA (modRNA) in human PSCs and showed that an addition of 5′UTR indeed enhanced protein expression. With the optimized modRNAs expressing ETV2 or ETV2 and GATA2, we are able to produce VE-cadherin(+) endothelial cells and CD34(+)CD43(+) hematopoietic progenitors, respectively, from human PSCs as well as non-human primate (NHP) PSCs. Overall, our findings provide valuable information on the design of in vitro transcription templates being used in PSCs and its broad applicability for basic research, disease modeling, and regenerative medicine.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43−CD73−DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs., It is unclear whether arterial specification is required for hematopoietic stem cell formation. Here, the authors use a chemically defined human pluripotent stem cell (hPSC) differentiation system to show the role of NOTCH signaling in forming arterial-type hemogenic endothelial cells.

Summary In this study, we demonstrate a newly derived mouse model that supports engraftment of human hematopoietic stem cells (HSCs) in the absence of irradiation. We cross the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) strain with the C57BL/6J-KitW-41J/J (C57BL/6.KitW41) strain and engraft, without irradiation, the resulting NBSGW strain with human cord blood CD34+ cells. At 12-weeks postengraftment in NBSGW mice, we observe human cell chimerism in marrow (97% ± 0.4%), peripheral blood (61% ± 2%), and spleen (94% ± 2%) at levels observed with irradiation in NSG mice. We also detected a significant number of glycophorin-A-positive expressing cells in the developing NBSGW marrow. Further, the observed levels of human hematopoietic chimerism mimic those reported for both irradiated NSG and NSG-transgenic strains. This mouse model permits HSC engraftment while avoiding the complicating hematopoietic, gastrointestinal, and neurological side effects associated with irradiation and allows investigators without access to radiation to pursue engraftment studies with human HSCs., Highlights • In engraftment experiments, nonirradiated NBSGW mice show enhanced humanization • Similar levels of human chimerism are observed between both irNSG and NBSGW mice • NBSGW mice are conducive to serial transplantation without irradiation • NBSGW mice harbor a mutant KitW41 allele, aiding Gly-A+ development in the marrow, In a newly generated murine strain, Thomson et al. demonstrate enhanced engraftment of human hematopoietic stem cells in the absence of irradiation. Maintained as a homozygous line, a mutant Kit (W41) allele, combined with Prkdcscid, Il2rγ−, and NOD.Sirpa, yields humanized grafts comparable to those observed in irradiated strains. This advancement allows researchers to humanize hosts without ionizing radiation.

Summary The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However, the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here, we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC), an extracellular matrix protein associated with HSC niches, strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders, including the formation of VE-Cadherin+CD73−CD235a/CD43− HE and hematopoietic progenitors with myeloid and T lymphoid potential., Graphical Abstract, Highlights • Hemogenic endothelium is generated in a completely defined xenogen-free system • The system reproduces all stages of hematopoietic development • Tenascin C enhances hematoendothelial development from pluripotent stem cells • Tenesacin C uniquely supports T cell specification, In this article, Slukvin and colleagues describe a feeder- and xenogene-free chemically defined platform for differentiating human pluripotent stem cells into definitive hematopoietic cells. They find that Tenascin C, a matrix protein associated with hematopoietic stem cell niches, strongly promotes hemogenic endothelium development and T lymphoid commitment in this system.

Significant advances in cellular reprogramming technologies and hematopoietic differentiation from human pluripotent stem cells (hPSCs) have already enabled the routine production of multiple lineages of blood cells in vitro and opened novel opportunities to study hematopoietic development, model genetic blood diseases, and manufacture immunologically matched cells for transfusion and cancer immunotherapy. However, the generation of hematopoietic cells with robust and sustained multilineage engraftment has not been achieved. Here, we highlight the recent advances in understanding the molecular and cellular pathways leading to blood development from hPSCs and discuss potential approaches that can be taken to facilitate the development of technologies for de novo production of hematopoietic stem cells.

Human induced pluripotent stem cells (hiPSCs) show great promise for obesity treatment as they represent an unlimited source of brown/brite adipose progenitors (BAPs). However, hiPSC-BAPs display a low adipogenic capacity compared to adult-BAPs when maintained in a traditional adipogenic cocktail. The reasons of this feature are unknown and hamper their use both in cell-based therapy and basic research. Here we show that treatment with TGFβ pathway inhibitor SB431542 together with ascorbic acid and EGF were required to promote hiPSCs-BAP differentiation at a level similar to adult-BAP differentiation. hiPSC-BAPs expressed the molecular identity of adult-UCP1 expressing cells (PAX3, CIDEA, DIO2) with both brown (ZIC1) and brite (CD137) adipocyte markers. Altogether, these data highlighted the critical role of TGFβ pathway in switching off hiPSC-brown adipogenesis and revealed novel factors to unlock their differentiation. As hiPSC-BAPs display similarities with adult-BAPs, it opens new opportunities to develop alternative strategies to counteract obesity.

Advances in cellular reprogramming technologies have created alternative platforms for the production of blood cells, either through inducing pluripotency in somatic cells or by way of direct conversion of nonhematopoietic cells into blood cells. However, de novo generation of hematopoietic stem cells (HSCs) with robust and sustained multilineage engraftment potential remains a significant challenge. Hemogenic endothelium (HE) has been recognized as a unique transitional stage of blood development from mesoderm at which HSCs arise in certain embryonic locations. The major aim of this review is to summarize historical perspectives and recent advances in the investigation of endothelial to hematopoietic transition (EHT) and HSC formation in the context of aiding in vitro approaches to instruct HSC fate from human pluripotent stem cells. In addition, direct conversion of somatic cells to blood and HSCs and progression of this conversion through HE stage are discussed. A thorough understanding of the intrinsic and microenvironmental regulators of EHT that lead to the acquisition of self-renewal potential by emerging blood cells is essential to advance the technologies for HSC production and expansion.

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study, we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34+CD43+ hematopoietic progenitors and CD31+CD43− endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43+ cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43+CD235a+CD41a± (erythro-megakaryopoietic), lin−CD34+CD43+CD45− (multipotent), and lin−CD34+CD43+CD45+ (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal, or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks.

Objective Cellular and molecular changes that occur during the genesis of the hematopoietic system and hematopoietic stem cells in the human embryo are mostly inaccessible to study and remain poorly understood. To address this gap we have exploited the human embryonic stem cell (hESC) system to molecularly characterize the global transcriptomes of the two functionally discreet and phenotypically separable populations of multipotent hematopoietic cells that first appear when hESCs are induced to differentiate on OP9 cells. Materials and Methods We prepared long serial analysis of gene expression libraries from lin − CD34 + CD43 + CD45 − and lin − CD34 + CD43 + CD45 + subsets of primitive hematopoietic cells derived in vitro from hESCs, sequenced them to a depth of 200,000 tags and compared their content with similar libraries prepared from highly purified populations of very primitive human fetal liver and cord blood hematopoietic cells. Results Comparison of libraries obtained from hESC-derived lin − CD34 + CD43 + CD45 − and lin − CD34 + CD43 + CD45 + revealed differences in their expression of genes associated with myeloid development, cellular biosynthetic processes, and cell-cycle regulation. Further comparisons with analogous data for primitive hematopoietic cells isolated from first-trimester human fetal liver and newborn cord blood showed an apparent similarity between the transcriptomes of the most primitive hESC- and in vivo–derived populations, with the main differences involving genes that regulate HSC self-renewal and homing, chromatin remodeling, AP1 transcription complex genes, and noncoding RNAs. Conclusion These data suggest that primitive hematopoietic cells are generated from hESCs in vitro by processes similar to those operative during human embryogenesis in vivo, although some differences were also detected.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

During hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile.

Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells, we were able to obtain up to 20% of CD34+ cells and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell–derived CD34+ cells were highly enriched in colony-forming cells, cells expressing hematopoiesis-associated genes GATA-1, GATA-2, SCL/TAL1, and Flk-1, and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90, CD117, and CD164, along with a lack of CD38 expression and contained aldehyde dehydrogenase–positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor, Flt3-L, interleukin 7 (IL-7), and IL-3, isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells.

We report a case of combined microcystic adenoma and pancreatic endocrine neoplasm of the pancreas in a 53-year-old male patient. The pancreatic tumor was an incidental computed tomography scan finding and was not accompanied by gastrointestinal symptoms. The tumor was located in the head of the pancreas and was composed of numerous small cysts lined by uniform clear cells with a centrally located solid endocrine component. Four cases of similar neoplasm have recently been reported, exclusively in women. Literature review and case analysis indicate that combined microcystic adenoma and pancreatic endocrine neoplasm is characterized by the presence of pancreatic endocrine neoplasm within microcystic adenoma in the head of the pancreas, affects women more often than men, and presents at a younger age when compared to microcystic adenoma.

SummaryEpigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in nonexpressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.

Hemogenic endothelium (HE) has been recognized as a source of hematopoietic stem cells (HSCs) in the embryo. Access to human HE progenitors (HEPs) is essential for enabling the investigation of the molecular determinants of HSC specification. Here, we show that HEPs capable of generating definitive hematopoietic cells can be obtained from human pluripotent stem cells (hPSCs) and identified precisely by a VE-cadherin(+)CD73(-)CD235a/CD43(-) phenotype. This phenotype discriminates true HEPs from VE-cadherin(+)CD73(+) non-HEPs and VE-cadherin(+)CD235a(+)CD41a(-) early hematopoietic cells with endothelial and FGF2-dependent hematopoietic colony-forming potential. We found that HEPs arise at the post-primitive-streak stage of differentiation directly from VE-cadherin-negative KDR(bright)APLNR(+)PDGFRα(low/-) hematovascular mesodermal precursors (HVMPs). In contrast, hemangioblasts, which are capable of forming endothelium and primitive blood cells, originate from more immature APLNR(+)PDGFRα(+) mesoderm. The demarcation of HEPs and HVMPs provides a platform for modeling blood development from endothelium with a goal of facilitating the generation of HSCs from hPSCs.

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.

Differentiation of human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into the erythroid lineage of cells offers a novel opportunity to study erythroid development, regulation of globin switching, drug testing, and modeling of red blood cell (RBC) diseases in vitro. Here we describe an approach for the efficient generation of RBCs from hiPSC/hESCs using an OP9 coculture system to induce hematopoietic differentiation followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. We showed that fibroblast-derived transgenic hiPSCs generated using lentivirus-based vectors and transgene-free hiPSCs generated using episomal vectors can be differentiated into RBCs with an efficiency similar to that of H1 hESCs. Erythroid cultures established with this approach consisted of an essentially pure population of CD235a(+)CD45(-) leukocyte-free RBCs with robust expansion potential and long life span (up to 90 days). Similar to hESCs, hiPSC-derived RBCs expressed predominately fetal γ and embryonic ɛ globins, indicating complete reprogramming of β-globin locus following transition of fibroblasts to the pluripotent state. Although β-globin expression was detected in hiPSC/hESC-derived erythroid cells, its expression was substantially lower than the embryonic and fetal globins. Overall, these results demonstrate the feasibility of large-scale production of erythroid cells from fibroblast-derived hiPSCs, as has been described for hESCs. Since RBCs generated from transgene-free hiPSCs lack genomic integration and background expression of reprogramming genes, they would be a preferable cell source for modeling of diseases and for gene function studies.

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.

Designer Stem Cells Despite their promise for use as disease models and in regenerative medicine, the generation of human-induced pluripotent stem (iPS) cells has been hindered by the integration of vector and transgenes in the host cell genome. Recent studies using the Cre/LoxP recombination strategy and the piggyBac transposon approach have approached this objective. However, Yu et al. (p. 797 , published online 26 March) now show the derivation of human iPS cells from postnatal foreskin fibroblasts using the nonintegrating oriP/EBNA1-based episomal vectors. The resultant iPS cells show characteristics of human embryonic stem cells and are free of vector and transgenes.

Embryonic stem cells represent a pluripotent population of cells capable of self-renewal, large-scale expansion, and differentiation in various cell lineages including cells of hematopoietic lineage. In this chapter, we describe a three-step cell culture method for directed differentiation of human embryonic stem cells (hESCs) to dendritic cells (DCs) that includes (1) hESC differentiation into hematopoietic progenitors by coculture with OP9 stromal cells, (2) expansion of myeloid DC precursors in suspension bulk cultures with granulocyte monocyte-colony stimulating factor (GM-CSF), and (3) differentiation of myeloid precursors to DCs in the serum-free medium with GM-CSF and interleukin-4 (IL-4). The method employs cell culture conditions selecting an almost pure population of myeloid DC precursors and does not require isolation of hematopoietic progenitors. With this method, hESCs can be differentiated to functional DCs within 30 days at an efficiency of at least four DCs per single undifferentiated hESC. Directed differentiation of DCs from hESCs could be useful for studying cellular and molecular mechanisms of DC development and potentially for the generation of antigen-presenting cells for cellular immunotherapy.

An important physiological function of vascular endothelial cells is to detect and respond to physical stimuli. While many efforts have been made to derive endothelial cells from human embryonic stem cells (hESCs), the ability of these derivatives to respond to mechanical forces has yet to be ascertained. hESC-derived endothelial cells (hEECs) were obtained by coculturing hESCs with OP9 stromal cells. Here we applied physiologic levels of shear stress to hEECs in a parallel plate flow chamber and observed changes in cell morphology and gene expression, comparing the response to that of human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs). Shear induced hEECs to elongate and align in the direction of flow, and their overall transcriptional response to shear was similar to the primary cells tested. In response to shear in hEECs, COX2 and MMP1 were upregulated four- and threefold, MCP1 and VCAM1 expression decreased over fivefold, and ICAM1 and TPA were downregulated almost threefold. TGFbeta1 and SOD2 transcription exhibited no change under the conditions tested. Additionally, preshearing of hEECs mitigated TNFalpha-induced VCAM1 surface expression. These findings suggest that hEECs are capable of functionally responding to changes in fluid shear stress by modulating gene expression and cell morphology.

Human embryonic stem cells (hESCs) represent a unique population of cells capable of self-renewal and differentiation into all types of somatic cells, including hematopoietic and endothelial cells. Since the pattern of hematopoietic and endothelial development observed in the embryo can be reproduced using ESCs differentiated in culture, hESCs can be used as a model for studies of specification and diversification of hematoendothelial progenitors. In addition, hESCs can be seen as a scalable source of hematopoietic and endothelial cells for in vitro studies. This unit describes a method for efficient differentiation of hESCs into hematopoietic progenitors and endothelial cells through coculture with mouse OP9 bone marrow stromal cells, as well as an approach for their analysis and isolation. Support protocols are provided for culture of mouse embryonic fibroblasts, evaluation of hematopoietic and endothelial differentiation by flow cytometry and colony-forming assay, removal of OP9 cells, and propagation of hESC-derived endothelial cells. Curr. Protoc.

Progress toward clinical application of ESC-derived hematopoietic cellular transplantation will require rigorous evaluation in a large animal allogeneic model. However, in contrast to human ESCs (hESCs), efforts to induce conclusive hematopoietic differentiation from rhesus macaque ESCs (rESCs) have been unsuccessful. Characterizing these poorly understood functional differences will facilitate progress in this area and likely clarify the critical steps involved in the hematopoietic differentiation of ESCs. To accomplish this goal, we compared the hematopoietic differentiation of hESCs with that of rESCs in both EB culture and stroma coculture. Initially, undifferentiated rESCs and hESCs were adapted to growth on Matrigel without a change in their phenotype or karyotype. Subsequent differentiation of rESCs in OP9 stroma led to the development of CD34+CD45− cells that gave rise to endothelial cell networks in methylcellulose culture. In the same conditions, hESCs exhibited convincing hematopoietic differentiation. In cytokine-supplemented EB culture, rESCs demonstrated improved hematopoietic differentiation with higher levels of CD34+ and detectable levels of CD45+ cells. However, these levels remained dramatically lower than those for hESCs in identical culture conditions. Subsequent plating of cytokine-supplemented rhesus EBs in methylcellulose culture led to the formation of mixed colonies of erythroid, myeloid, and endothelial cells, confirming the existence of bipotential hematoendothelial progenitors in the cytokine-supplemented EB cultures. Evaluation of four different rESC lines confirmed the validity of these disparities. Although rESCs have the potential for hematopoietic differentiation, they exhibit a pause at the hemangioblast stage of hematopoietic development in culture conditions developed for hESCs.

We have established a system for directed differentiation of human embryonic stem (hES) cells into myeloid dendritic cells (DCs). As a first step, we induced hemopoietic differentiation by coculture of hES cells with OP9 stromal cells, and then, expanded myeloid cells with GM-CSF using a feeder-free culture system. Myeloid cells had a CD4+CD11b+CD11c+CD16+CD123lowHLA-DR− phenotype, expressed myeloperoxidase, and included a population of M-CSFR+ monocyte-lineage committed cells. Further culture of myeloid cells in serum-free medium with GM-CSF and IL-4 generated cells that had typical dendritic morphology; expressed high levels of MHC class I and II molecules, CD1a, CD11c, CD80, CD86, DC-SIGN, and CD40; and were capable of Ag processing, triggering naive T cells in MLR, and presenting Ags to specific T cell clones through the MHC class I pathway. Incubation of DCs with A23187 calcium ionophore for 48 h induced an expression of mature DC markers CD83 and fascin. The combination of GM-CSF with IL-4 provided the best conditions for DC differentiation. DCs obtained with GM-CSF and TNF-α coexpressed a high level of CD14, and had low stimulatory capacity in MLR. These data clearly demonstrate that hES cells can be used as a novel and unique source of hemopoietic and DC precursors as well as DCs at different stages of maturation to address essential questions of DC development and biology. In addition, because ES cells can be expanded without limit, they can be seen as a potential scalable source of cells for DC vaccines or DC-mediated induction of immune tolerance.

Here, we examine the ability of undifferentiated human embryonic stem cells (hESCs) to reprogram the nuclei of hESC-derived myeloid precursors following cell–cell fusion. Using an OP9 coculture system, we produced CD45+ CD33+ myeloperoxidase+ myeloid precursors from an Oct4–enhanced green fluorescent protein (EGFP) knock-in hESC line and demonstrated that Oct4-EGFP expression was extinguished in these precursors. Upon fusion with undifferentiated hESCs, EGFP expression from the endogenous Oct4 promoter/regulatory region was re-established, ESC-specific surface antigens and marker genes were expressed, and myeloid precursor-specific antigens were no longer detectable. When the hybrid cells were formed into embryoid bodies, upregulation of genes characteristic of the three germ layers and extraembryonic tissues occurred, indicating that the hybrid cells had the potential to differentiate into multiple lineages. Interestingly, the hybrid cells were capable of redifferentiating into myeloid precursors with efficiency comparable with that of diploid hESCs despite their neartetraploid chromosome complement. These results indicate that hESCs are capable of reprogramming nuclei from differentiated cells and that hESC hybrid cells provide a new model system for studying the mechanisms of nuclear reprogramming.

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors ( OCT4, SOX2, NANOG , and LIN28 ) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.

SummaryAmong the three embryonic germ layers, the mesoderm is a major source of the mesenchymal precursors giving rise to skeletal and connective tissues, but these precursors have not previously been identified and characterized. Using human embryonic stem cells directed toward mesendodermal differentiation, we show that mesenchymal stem/stromal cells (MSCs) originate from a population of mesodermal cells identified by expression of apelin receptor. In semisolid medium, these precursors form FGF2-dependent compact spheroid colonies containing mesenchymal cells with a transcriptional profile representative of mesoderm-derived embryonic mesenchyme. When transferred to adherent cultures, individual colonies give rise to MSC lines with chondro-, osteo-, and adipogenic differentiation potentials. Although the MSC lines lacked endothelial potential, endothelial cells could be derived from the mesenchymal colonies, suggesting that, similar to hematopoietic cells, MSCs arise from precursors with angiogenic potential. Together, these studies identified a common precursor of mesenchymal and endothelial cells, mesenchymoangioblast, as the source of mesoderm-derived MSCs.