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Cellular therapies combined with genetic engineering technologies have emerged as increasingly powerful tools for immunotherapies of cancers and viral infections including HIV infection. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of blood progenitors and terminally differentiated cells can further expand applicability of cellular immunotherapies by offering “off-the-shelf” therapeutic products to fit specific clinical needs for a broad group of patients. In this review, we discuss current advances in lymphoid and myeloid cell differentiation of iPSCs along with challenges in de novo generation of hematopoietic stem cells (HSCs) and allogeneic iPSC products for “off-the-shelf” immunotherapies.

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 The retinal pigment epithelium (RPE)-choriocapillaris (CC) complex in the eye is compromised in age-related macular degeneration (AMD) and related macular dystrophies (MDs), yet in vitro models of RPE-CC complex that enable investigation of AMD/MD pathophysiology are lacking. By incorporating iPSC-derived cells into a hydrogel-based extracellular matrix, we developed a 3D RPE-CC model that recapitulates key features of both healthy and AMD/MD eyes and provides modular control over RPE and CC layers. Using this 3D RPE-CC model, we demonstrated that both RPE- and mesenchyme-secreted factors are necessary for the formation of fenestrated CC-like vasculature. Our data show that choroidal neovascularization (CNV) and CC atrophy occur in the absence of endothelial cell dysfunction and are not necessarily secondary to drusen deposits underneath RPE cells, and CC atrophy and/or CNV can be initiated systemically by patient serum or locally by mutant RPE-secreted factors. Finally, we identify FGF2 and matrix metalloproteinases as potential therapeutic targets for AMD/MDs.

The discovery that CCR5 serves as an R5-HIV-1 co-receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on genome editing of CCR5. Advancing translation of CCR5-mutant-based cellular therapies for HIV requires development of novel physiologically relevant animal models. Mauritian cynomolgus macaques (MCMs), with high degree of MHC allele sharing, are valuable models for HIV-1 research and stem cell therapies. To facilitate the generation of a CCR5-mutant MHC-defined MCM model, we explored editing the CCR5 gene in MCM embryos via CRISPR-Cas9. We refined ovarian stimulation and in vitro fertilization (IVF) methods established for Chinese cynomolgus macaques to generate in vitro MCM embryos. Time-lapse embryo imaging was performed to assess the timing of MCM embryonic developmental events in control and CRISPR-Cas9 microinjected embryos. Using a dual-guide gene targeting approach, biallelic deletions in the CCR5 gene were introduced into ~ 23–37% of MCM embryos. In addition, single blastomere PCR analysis revealed mosaicism in CCR5 editing within the same embryo. Successful development of IVF and CCR5 editing protocols in MCM embryos lays a foundation for the creation of CCR5-mutant MCMs to assess novel stem cell-based HIV therapeutics.

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.

Bone marrow suppression and associated neutropenia that renders the patient more susceptible to infection is a major complication and limiting factor for current chemotherapy. Strategies aimed at neutrophil reconstitution using transfusions or accelerating neutrophil recovery with G-CSF show a limited effect in reducing the rate of infection. To overcome these limitations, administration of ex vivo expanded somatic hematopoietic progenitors (HPs) has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival following myeloablation. Recent advances in reprogramming and induced pluripotent stem cell (iPSC) technologies have created alternative platforms for off-the-shelf supply of immunologically compatible HPs, including cellular products derived from MHC homozygous superdonors which can increase the degree of MHC matching and may provide a maximum utility for stem cell banking. In these studies, we developed Mauritian cynomolgus macaque (MCM) model to evaluate the utility and safety of CD34 +CD45 + hematopoietic progenitors derived from iPSCs (iHPs) generated from MHC homozygous animals in the treatment of cytopenia following myeloablative stem cell transplantation. MCM iPSCs were generated from MHC homozygous animals and used to generate iHPs in coculture with OP9. Three groups of MCMs underwent myeloablative total body irradiation (TBI) followed by transplantation of cryopreserved autologous CD34 + HSPCs. Four days after autologous HSPC transplantation, animals received transfusion of 30x10 6/kg cryopreserved iCD34 + cells tagged with eGFP or tdTomato from homozygous MHC-matched iPSCs (group 2) or homozygous MHC mismatched iPSCs (group 3). We have demonstrated that infusion of iHPs is safe and well tolerated. No teratoma or tumor formation was observed in animals one year after infusion of iHPs. Although we detected few iPSC derived hematopoietic cells in the blood within 1-week post-infusion, we did not see any significant differences in blood counts or other peripheral blood parameters between all animal groups. Intriguingly we found footprints of iPSC-derived cells in the colon, lymph node, skin and brain specimens collected 25 days after iHP infusion from one animal. This suggests a possibility of migration and retention within these tissues of iHP and iHP-derived myeloid cells. To assess immunogenicity, MHC homozygous iHPs were infused into immunocompetent MCMs across different MHC barriers. 10x10 6/kg of cryopreserved iCD34+ from homozygous iPSCs were transfused every week for 3 weeks, into animals that were divided into 4 groups namely autologous (group I), MHC matched homozygous (group II), MHC matched heterozygous (group III) and MHC mismatch (group IV). Overall, these studies revealed low immunogenicity of MHC homozygous iHPs in MHC matched homozygous and heterozygous animals, while weak immune response was detected following iHP infusion in some MHC mismatched animals. Disclosures Slukvin: Cynata Therapeutics: Consultancy, Current equity holder in publicly-traded company.

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

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.

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.

Wnt signaling pathway plays a major role in leukemogenesis of myeloid leukemia. Aberrancy in its regulation results in hyperactivity of the pathway contributing to leukemia propagation and maintenance. To investigate effects of Wnt pathway inhibition in leukemia, we used human leukemia cell lines (i.e., K562, HL60, THP1, and Jurkat) and several Wnt inhibitors, including XAV939, IWP2 and FH535. Our results showed that leukemia cell lines (>95 % cells) had increased endogenous levels of β-catenin as compared to mononuclear cells from healthy donors (0 %). Among the tested inhibitors, FH535 demonstrated a markedly suppressive effect (IC50 = 358 nM) on mRNA levels of β-catenin target genes (LEF1, CCND1, and cMYC). In addition, FH535 significantly potentiated imatinib-induced apoptosis. Evaluation of erythrocyte and megakaryocyte lineage using flow cytometry demonstrated that the potentiation mechanism is independent of the developmental stage, and is more likely due to crosstalk between other pathways and β-catenin. FH535 also displayed antiproliferative properties in other cell lines used in this study. In summary, FH535 showed significantly high antiproliferative effects at submicromolar dosages, and additionally enhanced imatinib-induced apoptosis in human leukemia cell lines. Our results highlight its potential antileukemic promise when used in conjunction with other conventional therapeutic regimens.

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.

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.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

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.

Human pluripotent stem cells (hPSCs) offer the potential to serve as a versatile and scalable source of T cells for immunotherapies, which could be coupled with genetic engineering technologies to meet specific clinical needs. To improve T cell production from hPSCs, it is essential to identify cell subsets that are highly enriched in T cell progenitors and those stages of development at which NOTCH activation induces the most potent T cells. In this study, we evaluated the efficacy of T cell production from cell populations isolated at different stages of hematopoietic differentiation, including mesoderm, hemogenic endothelium (HE), and multipotent hematopoietic progenitors. We demonstrate that KDRhiCD31− hematovascular mesodermal progenitors (HVMPs) with definitive hematopoietic potential produce the highest numbers of T cells when cultured on OP9-DLL4 as compared with downstream progenitors, including HE and multipotent hematopoietic progenitors. In addition, we found that T cells generated from HVMPs have the capacity to expand for 6–7 wk in vitro, in comparison with T cells generated from HE and hematopoietic progenitors, which could only be expanded for 4–5 wk. Demonstrating the critical need of NOTCH activation at the HVMP stage of hematopoietic development to establish robust T cell production from hPSCs may aid in establishing protocols for the efficient off-the-shelf production and expansion of T cells for treating hematologic malignancies.

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 Advances in the scalable production of blood cells from induced pluripotent stem cells (iPSCs) open prospects for the clinical translation of de novo generated blood products, and evoke the need for preclinical evaluation of their efficacy, safety, and immunogenicity in large animal models. Due to substantial similarities with humans, the outcomes of cellular therapies in non-human primate (NHP) models can be readily extrapolated to a clinical setting. However, the use of this model is hampered by relatively low efficiency of blood generation and lack of lymphoid potential in NHP-iPSC differentiation cultures. Here, we generated transgene-free iPSCs from different NHP species and showed the efficient induction of mesoderm, myeloid, and lymphoid cells from these iPSCs using a GSK3β inhibitor. Overall, our studies enable scalable production of hematopoietic progenitors from NHP-iPSCs, and lay the foundation for preclinical testing of iPSC-based therapies for blood and immune system diseases in an NHP model., Graphical Abstract, Highlights • Generation of transgene-free iPSCs from various non-human primate (NHP) species • GSK3β inhibition is essential for induction of mesoderm and blood from NHP-iPSCs • Efficient generation of CD34+CD45+CD90+CD38−CD45RA− progenitors from NHP-iPSCs • Efficient T and NK cell production from NHP-iPSCs, In this article, Slukvin and colleagues show that the Wnt agonist CHIR99021 promotes efficient myeloid and lymphoid hematopoiesis from induced pluripotent stem cells (iPSCs) generated from various non-human primate (NHP) species. These studies enable scalable production of hematopoietic progenitors from NHP-iPSCs, and lay the foundation for preclinical testing of iPSC-based therapies for blood and immune system diseases in an NHP model.

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.

Recent advances in understanding the major bottlenecks in derivation of engraftable HSCs and lymphoid cells from pluripotent stem cells (PSC), have identified deficiencies in NOTCH and HOXA signaling as contributing factors to the observed functional deficits of PSC-derived hematopoietic progenitors. However, little is known about the mechanisms that are essential for establishing these pathways during PSC differentiation. Here, we revealed the critical role of SOX17 in linking HOXA and NOTCH-mediated arterial programs in hemogenic endothelium (HE) and specification of definitive lympho-myeloid hematopoiesis. Using SOX17-knockout (SOX17-/-) and SOX17 DOX-inducible (iSOX17) hESCs, we found that SOX17-deficiency substantially reduces formation of CD144+CD43-CD73-DLL4+CXCR4+/- arterial HE and definitive lympho-myeloid hematopoiesis, while SOX17 upregulation at mesodermal stage of development causes the opposite effect. Molecular profiling of HE generated from iSOX17 hESCs in DOX+ and DOX- conditions using RNAseq, SOX17 ChIPseq and ATACseq, revealed that SOX17 overexpression upregulates 522 genes enriched in NOTCH, TGFb, HEDGEHOG and WNT signaling, including DLL1, DLL4, NOTCH4, LFNG, WNT5a, WNT5b, GLI3, and genes associated with HSC development, CXCR4,KITLG and ALDH1A2. In addition, we noted significant upregulation of HOXA7,HOXA9, HOXA10, HOXB8, HOXC4 and CDX2 homeobox genes in SOX17-induced cultures, with no expression of HOXA genes observed in HE from SOX17-/- cells. ChIPSeq analysis revealed DOX+ specific SOX17 binding at transcriptional start sites (TSS) of 316 significantly upregulated genes, including ALDH1A2, CDX2, DLL1, DLL4, HEY1, HOXA7, HOXB8, HOXC4 and KITLG, suggesting that upregulation of these genes could be explained by their direct activation by SOX17. Since ALDH1A2 and CDX2 are known to play a role in the activation of HOXA genes, we investigated whether SOX17's effect on HOXA expression could also be mediated by ALDH1A2 and CDX2. We found that adding ALDH1 inhibitor to DOX+ cultures had no effect on arterial HE development and HOXA expression. In contrast, transfection of iSOX17 hPSCs cultures with CDX2 shRNA significantly decreased arterial HE formation and downregulated HOXA7, HOXA9, and HOXA10 expression. Overall, our studies indicate that SOX17 plays a critical role in the activation and integration of arterial and HOXA programs in HE, which is mediated by CDX2. These findings will be important for designing a strategy for direct HSC fate programming from hPSCs. Disclosures Uenishi: Casebia Therapeutics: Employment. Slukvin:Cynata Therapeutics: Consultancy, Other: Founder and Stockholder.

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.

Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.

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.

Human pluripotent stem cell (hPSCs) have created alternative platforms for producing blood cells for transfusion, immunotherapies, and transplantation. Advancing blood cell manufacturing from hPSCs and translating hPSC-based technologies to the clinic requires improving the scalabilty of blood cell production through enhancing hematopoietic differentiation from hPSCs, and increasing expansion of lineage committed hematopoietic progenitors. The pyrimido-indole derivative UM171 has been described as one the most potent small molecules agonists for HSC expansion in vitro. However, the effect and mechanism of UM171 action on hPSC-derived hematopoietic progenitors (HPs) has not been explored. It also remains unclear whether UM171 selectively expands the most primitive multipotential HPs with lin-CD34+CD43+HSC phenotype, or affects progenitors already committed to a particular hematopoietic cell lineage. In our studies, we evaluated the effect of UM171 on the expansion and differentiation of CD34+CD43+HPs that were generated in chemically defined, serum- and feeder-free conditions from hPSCs. We revealed that culture of hPSC-derived HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3, IL3 and IL6) in the presence of UM171 selectively expanded HPs with a unique CD34+CD41loCD45+phenotype. Isolation of this population by FACS revealed that it mostly possesses myeloid potential and is highly enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4 in presence of SCF, Flt-3 and IL7, UM171 predominately expands CD34+CD7+CD41a-lymphoid progenitors with NK cell potential and increases up to 10-fold NK cell output in NK differentiation cultures. NK cells generated with UM171 possess strong cytotoxicity, express perforin and upregulate IFNg production, following stimulation with K562 or PMA. As determined by annexin V immunostaining, UM171 treatment decreased the number of apoptotic HPs in expansion cultures. In addition, UM171 expansion of HPs was associated with increased proliferation, as determined by BrdU assay and Ki67 staining. Extending these observations, cell cycle analysis revealed that UM171 predominantly increases the proportion of HPs in the early S phase of the cell cycle. 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. Disclosures Slukvin: Cynata Therapeutics Limited: Consultancy, Equity Ownership.

Human pluripotent stem cells (hPSCs) emerged as an important tool to investigate human development and disease. These studies often require genetically engineering hPSCs to stably express a transgene, which remains functional in various hPSC progeny. PiggyBac transposon is a highly effective and technically simple vector system with large cargo space available for permanent gene delivery. This unit describes the use of PiggyBac transposons to genetically engineer hPSCs to introduce conditionally expressed transgene or reporter to effectively monitor gene expression during differentiation. Both methods enable robust generation of stable hPSC lines within 1 month. © 2018 by John Wiley & Sons, Inc.

Human embryonic stem cells (hESCs) offer the opportunity to create a novel source of blood cells for transfusion, transplantation and cancer immunotherapy. Identification of sequential progenitors leading to blood development, as well as a detailed understanding of the molecular mechanisms of hematopoietic lineage specification and diversification from hESCs, will be critical to advance technologies for large-scale production of blood cells and in vitro generation of hematopoietic stem cells. Multiple lines of evidence suggest that hematopoiesis, both in vivo during embryogenesis and in vitro from hESCs, is initiated from hemangioblasts; cells with the potential to generate both hematopoietic and endothelial cells. However, the phenotypic and functional properties of hemangioblasts remain largely unknown. The paper from Zambidis et al. is the first demonstration that hemangioblasts generated from hESCs express angiotensin-converting enzyme (CD143). More importantly, the current study demonstrates that the renin-angiotensin system plays a critical role in the hemangioblast fate decision to produce either blood or endothelial cells. These findings could be exploited for developing novel cellular and drug therapies for hematological and vascular diseases.

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.

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

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.

Leukocyte Ig-like receptors (LILRs) are a family of receptors that have inhibitory and activating functions and widely expressed by lymphoid and myeloid cells. Here we report the identification of the rhesus monkey LILRs by screening of rhesus spleen and decidua cDNA libraries and RT-PCR cloning. We obtained eight different full-length clones with structural and functional diversity similar to human LILRs, including LILRs with immunoreceptor tyrosine-based inhibitory motifs, LILRs with truncated cytoplasmic tails containing positively charged arginine residues in the transmembrane domain, and putative soluble receptors lacking transmembrane or cytoplasmic domains. Characterization of rhesus LILRs will facilitate use of this non-human primate model for the study of the functional role(s) of LILRs, including immune regulation through interaction with non-classical MHC class I molecules.

We performed immunohistochemical analysis of DC-SIGN expression at the maternal–fetal interface at different stages of pregnancy in the rhesus monkey. Natural killer cells, monocytes and macrophages were observed in the nonpregnant endometrium, particularly in the luteal phase, and were increased in pregnant endometrium. No DC-SIGN + cells were observed in the nonpregnant uterus. We observed decidual DC-SIGN + cells within 1 week of implantation, and they increased in number during the first 5 weeks of gestation. DC-SIGN + cells showed a clear differential distribution in the decidua in the first 2 weeks of pregnancy, being found only adjacent to the implantation site, in marked contrast to the widespread distribution of CD68 + macrophages and CD56 + NK cells throughout the endometrium. DC-SIGN + cells also showed a more dendritic morphology than the general CD68 + cell population, and analysis of serial sections indicated an overlapping but not identical localization of these markers. Mature dendritic cells could not be detected as judged by total absence of immunostaining for CD83, CD86, DEC-205, or CD1a. DC-SIGN + cells were defined as MHC class II + and CD14 + by flow cytometry. We conclude that DC-SIGN expression is an early response by the primate maternal immune system to the implanting embryo. The selectively distributed population of DC-SIGN + decidual leukocytes may represent a morphologically and phenotypically distinct subpopulation of decidual macrophages of early pregnancy that could contribute to the establishment of maternal–fetal immune tolerance.

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.

A major obstacle to the success of gene therapy for hematologic disorders is the inefficiency of lentiviral vector (LV) gene transfer to hematopoietic stem cells (HSCs). Achieving clinically relevant gene delivery levels requires prolonged ex vivo culture of HSCs with cytokines and large LV doses. Rapamycin, PGE2, and other small molecules, have been reported to increase LV transduction of HSCs, however the mechanism of action of these drugs and the basis for LV restriction in HSCs are poorly understood.Here, we report a novel small molecule, β-deliverin, which improves LV gene transfer to HSCs up to 3 fold over DMSO control in vitro. This effect is most pronounced in human adult peripheral blood-derived HSCs, but also observed in human cord-derived HSCs, and in non-human primate bone marrow-derived HSCs. Importantly, treatment with β-deliverin does not significantly affect the viability or expansion of HSCs in culture. Furthermore, for cord-derived HSCs, there was no reduction in the number or type of colony-forming cells. In vivo, β-deliverin treated HSCs engraft in the peripheral blood of NSG mice at levels comparable to control at 16 weeks post-engraftment. Despite an only modest increase in transduction efficiency in β-deliverin-treated cord-derived HSCs transduced in vitro (26% versus 20% for control), the marking frequency for control cells dropped to

A case of neonatal-onset glutaric acidemia type II with electron-transfer flavoprotein (ETF) deficiency is presented. The morphological pattern of disease in the male infant included hypospadias, cryptorchidism, bilateral 13 ribs, nuclear cataract, cystic dysplasia of kidneys, lipid accumulation in the liver and renal tubular epithelium, and immature brain with white matter gliosis. The morphologic examination of the placenta revealed features of delayed maturation, including large-for-gestational-age size and abundant immature intermediate villi with cellular syncytiotrophoblast, persistent villous cytotrophoblast, and decreased syncytial knots. In addition, immature intermediate villi showed exaggeration of lacunar interstitial spaces consistent with nonhydropic villous edema. Marked lipid accumulation was seen within extravillous trophoblasts of placental septa and cell islands. Light lipid accumulation was also noted within fibroblasts of stem villi. These findings suggest that in glutaric acidemia type II, fatty acid oxidation could also be affected in the placenta.

In this study, we carried out a phenotypic and functional characterization of lymphocytes isolated from the uterine endometrium of the pregnant rhesus monkey. A majority (80%) of these cells were CD56 bright+ , CD3− had typical large granular lymphocyte/uterine natural killer (NK) cell morphology and contained numerous cytoplasmic granules. Flow cytometric evaluation showed that rhesus decidual CD56 bright+ cells shared other phenotypic features of human uterine NK cells, including low levels of CD45RA and CD62L expression. A majority of the rhesus uterine CD56 bright+ cells expressed low levels of CD 16 but were CD2−. In contrast, most rhesus CD16+ peripheral blood cells were CD56−. In addition to the primary population of CD56 bright+ cells, a minor subset of smaller and less granular CD56 intermediate+ decidual lymphocytes was identified, the majority of which were CD16−, CD2 + . Decidual CD56+ cells did not express monocyte/macrophage markers, including CD14, CD64 and CD68. Decidual lymphocytes effectively lysed K562, Raji and particularly 721.221 targets in cytotoxicity assays. Together, these results suggest that as in human pregnancy, rhesus decidual CD56 bright+ cells represent a distinct lymphocyte subset that belongs to the NK cell lineage.

The rhesus macaque is an important animal model for several human diseases and organ transplantation. Therefore, definition of the MHC of this species is crucial to the development of these models. Unfortunately, unlike humans, lymphocytes from a single rhesus macaque express up to 12 different MHC class I cDNAs. From which locus these various alleles are derived is unclear. In our attempts to define the MHC class I loci of the rhesus macaque, we have identified an unusual MHC class I locus, Mamu-I. We isolated 26 I locus alleles from three different macaque species but not from three other Cercopithecine genera, suggesting that the I locus is the result of a recent duplication of the B locus occurring after the divergence of macaques from the ancestor of the other extant Cercopithecine genera. Mamu-I mRNA transcripts were detected in all tissues examined and Mamu-I protein was produced in rhesus B lymphoblastoid cell lines. Furthermore, Mamu-I protein was detected by flow cytometry on the surface of human 721.221 cells transfected with Mamu-I. In contrast to the polymorphism present at this locus, there is unusually low sequence variability, with the mean number of nucleotide differences between alleles being only 3.6 nt. Therefore, Mamu-I is less variable than any other polymorphic MHC class I locus described to date. Additionally, no evidence for positive selection on the peptide binding region was observed. Together, these results suggest that Mamu-I is an MHC class I locus in primates that has features of both classical and nonclassical loci.

We identified recently a novel major histocompatibility complex (MHC) class I locus in the rhesus monkey, Mamu-AG , which is expressed in the placenta and encodes molecules that share unique characteristics of human HLA-G. We established locus-specific reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assays to determine whether Mamu-AG is expressed in other rhesus monkey tissues. With an RT-PCR assay, Mamu-AG mRNA was detected in placenta, amniotic membranes, kidney, spleen, eye, brain, lung, spinal cord, liver and occasionally heart, but was undetectable in lymph nodes, salivary glands, peripheral blood lymphocytes (PBL), large and small intestine, skeletal muscle or skin. Examination of endocrine organs demonstrated the presence of Mamu-AG transcripts in pituitary, testes, ovary and adrenal glands but not in pancreas or thyroid. Quantitative analysis using a ribonuclease protection assay demonstrated that the highest level of Mamu-AG mRNA expression was consistently in the placenta and amniotic membranes, while expression was moderate in a few tissues (testis, adrenal) and low to undetectable in all other tissues. These results suggest that the Mamu-AG mRNA, like the mRNA for the human MHC class Ib gene HLA-G, is expressed at high levels in the placenta, but also has restricted low-level expression in other tissues.

The human placenta expresses the nonclassical major histo-compatibility complex (MHC) class I molecule, human lymphocyte antigen (HLA)-G, which may contribute to the establishment of maternal-fetal immune tolerance. Although the HLA-G ortholog of the rhesus monkey, Mamu-G, is a pseudogene, another nonclassical MHC class I locus, Mamu-AG, is expressed in the rhesus monkey placenta. Mamu-AG encodes MHC class I A locus-related molecules that exhibit all the characteristics of human HLA-G, including limited polymorphism and a truncated cytoplasmic domain. We have examined MHC class I glycoprotein and Mamu-AG mRNA expression in the rhesus placenta and in cultured trophoblasts. Immunocytochemical analysis of rhesus placental tissues with the W6/32 monoclonal antibody demonstrated a high level of MHC class I expression in villous syncytiotrophoblasts, whereas villous cytotrophoblasts were largely MHC class I negative. Only low levels of MHC class I expression were seen in extravillous cytotrophoblasts of cell columns and the trophoblastic shell. In situ hybridization demonstrated that Mamu-AG mRNAs were expressed at a high level in first-trimester villous syncytiotrophoblasts. MHC class I and Mamu-AG expression was significantly up-regulated during in vitro culture and differentiation of freshly isolated villous cytotrophoblasts into syncytiotrophoblasts. Preferential Mamu-AG expression in syncytiotrophoblasts suggests that rhesus monkey MHC class I-bearing trophoblasts could potentially interact with maternal peripheral blood lymphocytes rather than with uterine decidual lymphocytes as has been proposed for human trophoblasts.

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.

Human induced pluripotent stem cells (iPSCs) have become an important tool for modeling human diseases and are considered a potential source of therapeutic cells. Original methods for iPSC generation use fibroblasts as a cell source for reprogramming and retroviral vectors as a delivery method of the reprogramming factors. However, fibroblasts require extended time for expansion and viral delivery of transgenes results in the integration of vector sequences into the genome which is a source of potential insertion mutagenesis, residual expressions, and reactivation of transgenes during differentiation. Here, we provide a detailed protocol for the efficient generation of transgene-free iPSC lines from human bone marrow and cord blood cells with a single transfection of non-integrating episomal plasmids. This method uses mononuclear bone marrow and cord blood cells, and makes it possible to generate transgene-free iPSCs 1-3 weeks faster than previous methods of reprogramming with fibroblasts. Additionally, we show that this approach can be used for efficient reprogramming of chronic myeloid leukemia cells.

The effects of ethanol (EtOH) consumption by adult female C57B1/6 mice on lymphocyte populations of the mesenteric lymph nodes (MLNs) were determined by feeding mice with the Lieber-DeCarli liquid diet by a pair-feeding paradigm. Histological analysis of the MLNs of EtOH-fed mice showed a progressive loss of lymphocytes from the medullary regions at 3, 5, and 7 days after initiation of the EtOH diet. The stromal cells in the medullary region also demonstrated a progressive alteration in stellate morphological features at times corresponding to those of loss of lymphocytes from this region. Microscopic evaluation of the follicle regions of MLNs obtained from mice fed an EtOH-containing diet showed no appreciable alterations in morphological characteristics. The number of tingible body macrophages in the germinal centers of the follicles, however, was increased after 3 days of EtOH diet feeding and declined progressively after this time. Flow cytometric analysis of isolated lymphocytes showed a depletion of both T and B cell populations from the MLNs. In contrast to B cells, however, T cells were depleted through 7 days of EtOH diet feeding. Total RNA isolated from the MLNs of mice consuming the EtOH-containing diet was progressively degraded. No degradation of DNA was observed. These study results establish that continuous consumption of dietary EtOH adversely affects the cellularity of MLN, resulting in a progressive loss of lymphocytes that is associated with degradation of total RNA.

Human induced pluripotent stem cells (hiPSCs) have become an important tool for the studies of human cell specification, disease modeling, and drug screening. They also hold promise for regenerative medicine as a potential source of patient-specific donor cells. Human cord blood (CB) represents one of the most accessible sources of somatic cells, with extensive and diverse archived samples available. The successful reprogramming of umbilical CB cells stored for more than 20 years suggests a minimal effect of storage on their properties, and opens these stocks for future exploration. Although isolated stem cells/progenitors and the viral delivery of reprogramming factors were utilized in the initial CB reprogramming methods, recent studies have demonstrated the efficient generation of hiPSCs free of exogenous sequences from just 1 ml of CB, using the unfractionated total mononuclear cells and the non-integrating Epstein–Barr virus-based episomal vectors with only one round of transfection. Advancing CB-derived hiPSCs towards clinical applications will require the development of completely defined conditions for hiPSC derivation and differentiation, and an evaluation of the therapeutic efficiency and safety of these cells in animal models. Keywords: Pluripotent stem cells; Embryonic stem cells; Induced pluripotent stem cells; Umbilical cord blood