Vincenzo Calvanese

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

GPI-80 Defines Self-Renewal Ability in Hematopoietic Stem Cells during Human Development

Advances in pluripotent stem cell and reprogramming technologies have given us the hope of generating hematopoietic stem cells (HSCs) in culture. To succeed, greater understanding of the self-renewing HSC during human development is required. We discovered that the glycophosphatidylinositol-anchored surface protein GPI-80 defines a subpopulation of human fetal liver hematopoietic stem/progenitor cells (HSPCs) with self-renewal ability. CD34(+)CD38(lo/-)CD90(+)GPI-80(+) HSPCs were the sole population that maintained proliferative potential and an undifferentiated state in stroma coculture and engrafted in immunodeficient mice. GPI-80 expression also enabled tracking of HSPCs once they emerged from endothelium and migrated between human fetal hematopoietic niches. GPI-80 colocalized on the surface of HSPCs with Integrin alpha-M (ITGAM), which in leukocytes cooperates with GPI-80 to support migration. Knockdown of GPI-80 or ITGAM was sufficient to compromise HSPC expansion in culture and engraftment in vivo. These findings indicate that human fetal HSCs employ mechanisms used in leukocyte adhesion and migration to mediate HSC self-renewal.

Medial HOXA genes demarcate haematopoietic stem cell fate during human development

Pluripotent stem cells (PSCs) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had the CD34+CD38−/loCD90+CD45+GPI-80+ fetal liver (FL) HSPC immunophenotype, but exhibited poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, medial HOXA gene expression induced by retinoic acid signalling marks the establishment of the definitive HSPC fate and controls HSPC identity and function.

Protagonist or antagonist? The complex roles of retinoids in the regulation of hematopoietic stem cells and their specification from pluripotent stem cells

Hematopoietic stem cells (HSCs) are multipotent cells responsible for the maintenance of the hematopoietic system throughout life. Dysregulation of the balance in HSC self-renewal, death, and differentiation can have serious consequences such as myelodysplastic syndromes or leukemia. All-trans retinoic acid (ATRA), the biologically active metabolite of vitamin A/RA, has been shown to have pleiotropic effects on hematopoietic cells, enhancing HSC self-renewal while also increasing differentiation of more mature progenitors. Furthermore, ATRA has been shown to have key roles in regulating the specification and formation of hematopoietic cells from pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Here, we summarize the known roles of vitamin A and RA receptors in the regulation of hematopoiesis from HSCs, ES, and iPSCs.

Sex hormone drives blood stem cell reproduction

Stem cells ensure the maintenance of tissue homeostasis throughout life by tightly regulating their self‐renewal and differentiation. In a recent study published in Nature, Nakada et al, 2014 unveil an unexpected endocrine mechanism that regulates hematopoietic stem cell (HSC) self‐renewal.

Tracking HSC Origin: From Bench to Placenta

Reporting in Developmental Cell, Pereira et al. (2016) use in vitro lineage reprogramming insights to inform understanding of hematopoietic stem cell (HSC) development in vivo. They find Prom1(+)Sca1(+)CD34(+)CD45(-) hemogenic precursors, akin to fibroblast-derived hemato-vascular precursors, in mouse placenta and embryo. The cells mature into transplantable HSCs in culture.