During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT). To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34 cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context.

BackgroundMesenchymal stromal cells (MSCs) are currently being evaluated in numerous pre-clinical and clinical cell-based therapy studies. Furthermore, there is an increasing interest in exploring alternative uses of these cells in disease modelling, pharmaceutical screening, and regenerative medicine by applying reprogramming technologies. However, the limited availability of MSCs from various sources restricts their use. Term amniotic fluid has been proposed as an alternative source of MSCs. Previously, only low volumes of term fluid and its cellular constituents have been collected, and current knowledge of the MSCs derived from this fluid is limited. In this study, we collected amniotic fluid at term using a novel collection system and evaluated amniotic fluid MSC content and their characteristics, including their feasibility to undergo cellular reprogramming.MethodsAmniotic fluid was collected at term caesarean section deliveries using a closed catheter-based system. Following fluid processing, amniotic fluid was assessed for cellularity, MSC frequency, in-vitro proliferation, surface phenotype, differentiation, and gene expression characteristics. Cells were also reprogrammed to the pluripotent stem cell state and differentiated towards neural and haematopoietic lineages.ResultsThe average volume of term amniotic fluid collected was approximately 0.4 litres per donor, containing an average of 7 million viable mononuclear cells per litre, and a CFU-F content of 15 per 100,000 MNCs. Expanded CFU-F cultures showed similar surface phenotype, differentiation potential, and gene expression characteristics to MSCs isolated from traditional sources, and showed extensive expansion potential and rapid doubling times. Given the high proliferation rates of these neonatal source cells, we assessed them in a reprogramming application, where the derived induced pluripotent stem cells showed multigerm layer lineage differentiation potential.ConclusionsThe potentially large donor base from caesarean section deliveries, the high yield of term amniotic fluid MSCs obtainable, the properties of the MSCs identified, and the suitability of the cells to be reprogrammed into the pluripotent state demonstrated these cells to be a promising and plentiful resource for further evaluation in bio-banking, cell therapy, disease modelling, and regenerative medicine applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0582-6) contains supplementary material, which is available to authorized users.

SummaryThe functions of retinoic acid (RA), a potent morphogen with crucial roles in embryogenesis including developmental hematopoiesis, have not been thoroughly investigated in the human setting. Using an in vitro model of human hematopoietic development, we evaluated the effects of RA signaling on the development of blood and on generated hematopoietic progenitors. Decreased RA signaling increases the generation of cells with a hematopoietic stem cell (HSC)-like phenotype, capable of differentiation into myeloid and lymphoid lineages, through two separate mechanisms: by increasing the commitment of pluripotent stem cells toward the hematopoietic lineage during the developmental process and by decreasing the differentiation of generated blood progenitors. Our results demonstrate that controlled low-level RA signaling is a requirement in human blood development, and we propose a new interpretation of RA as a regulatory factor, where appropriate control of RA signaling enables increased generation of hematopoietic progenitor cells from pluripotent stem cells in vitro.

Cell stressors, such as elevated levels of reactive oxygen species (ROS), adversely affect hematopoietic stem cell (HSC) reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human pluripotent stem cells (hPSCs). Using our previously described in vitro system for efficient derivation of hematopoietic cells from hPSCs, we show that the vast majority of generated hematopoietic cells display supraphysiological levels of ROS compared to fresh cord blood cells. Elevated ROS resulted in DNA damage of the CD34 1 hematopoietic fraction and, following functional assays, reduced colony formation and impaired proliferative capacity. Interestingly, all the proliferative potential of the most primitive hematopoietic cells was limited to a small fraction with low ROS levels. We show that elevation of ROS in hPSC-derived hematopoietic cells is contributed by multiple distinct cellular processes. Furthermore, by targeting these molecular processes with 4 unique factors, we could reduce ROS levels significantly, yielding a 22-fold increase in the most primitive CD90 1 CD34 1 hematopoietic cells with robust growth capacity. We demonstrate that the ROS reducing factors specifically reduced ROS in more primitive hematopoietic fractions, in contrast to endothelial cells that maintained low ROS levels in the cultures. We conclude that high levels of ROS in in vitro differentiation systems of hPSCs is a major determinant in the lack of ability to generate hematopoietic cells with similar proliferation/differentiation potential to in vivo hematopoietic progenitors, and suggest that elevated ROS is a significant barrier to generating hPSC-derived repopulating HSCs. STEM CELLS 2017;35:197-206 SIGNIFICANCE STATEMENTCell stressors, such as elevated levels of reactive oxygen species (ROS), adversely affect hematopoietic stem cell reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human pluripotent stem cells (hPSCs). Our results show for the first time that high levels of ROS, resulting from mechanisms associated with in vitro hPSC differentiation systems, occurs and functionally impairs the most primitive of the newly generated hematopoietic cells, and that ROS regulation is a requirement for generating functional primitive hematopoietic cells in vitro. We provide strong evidence that efforts to further normalize ROS toward physiological levels will be crucial to generate functionally equivalent human hematopoietic stem and progenitor cells in vitro as compared to their in vivo counterparts.

ABSTRACT. SMAP2 is an Arf GTPase-activating protein that is located and functions on early endosome membranes. In the present study, the trans-Golgi network (TGN) was verified as an additional site of SMAP2 localization based on its co-localization with various TGN-marker proteins. Mutation of specific stretches of basic amino acid residues abolished the TGN-localization of SMAP2. Over-expression of wild-type SMAP2, but not of the mutated SMAP2, inhibited the transport of vesicular stomatitis virus-G protein from the TGN to the plasma membrane. In contrast, this transport was enhanced in SMAP2 (-/-) cells characterized by increased levels of the activated form of Arf. SMAP2 therefore belongs to an ArfGAP subtype that resides on the TGN and functions as a negative regulator of vesicle budding from the organelle.