Hematopoietic specification from human pluripotent stem cells: current advances and challenges toward de novo generation of hematopoietic stem cells

Abstract

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.

Figure 1

Therapeutic potential of hPSCs for blood diseases. iPSCs can be potentially used to treat patients with monogenic genetic blood diseases such as sickle cell anemia, β-thalassemia, Fanconi anemia, or SCID (upper panel). Autologous skin or blood cells from these patients can be reprogrammed into iPSCs. The defective gene in iPSCs can be repaired using homologous recombination. De novo generation of HSCs from gene-corrected iPSCs would provide immunologically matched cells for bone marrow transplantation. For cancer therapy, autologous iPSCs could be generated from skin fibroblasts or other somatic cells lacking leukemia mutation and used to generate HSCs for bone marrow transplantation as well as immune cells to induce an anti-leukemia immune response (lower panel). Professional illustration by Paulette Dennis.

Figure 2

A model of hematopoietic development from hPSCs. (A) The most critical factors involved in specification of hematovascular precursors from PSCs and regulation of blood formation from HE. (B) Stages of hematopoietic development from hPSCs. Mesodermal stage of development is defined as expression of the mesodermal markers, APLNR and KDR.^,, The lack of expression of typical endothelial (CD31, VE-cadherin), endothelial/mesenchymal (CD73, CD105), and hematopoietic (CD43, CD45) markers, ie, ^EMHlin⁻ phenotype, separates mesoderm from lineage-committed cells.^, The most primitive mesodermal precursors with hematopoietic potential arise in coculture with OP9 or an embryoid body system on day 3 of differentiation. These cells have features of a posterior PS, coexpress KDR, APLNR, and PDGFRα and capable of forming BL (hemangioblast) colonies in the presence of FGF2 and VEGF.^,,, The formation of BL colonies in clonogenic medium proceeds through VE-cadherin⁺ endothelial intermediates, which generate primitive hematopoietic cells with erythroid, megakaryocytic, and macrophage potentials.^, Progressive mesodermal commitment to endothelial and hematopoietic cells is associated with downregulation of PDGFRα^, and PS genes, and upregulation of KDR, TAL1, and GATA2 genes associated with angiohematopoietic development leading to formation of ^EMHlin⁻KDR^brightAPLNR⁺PDGFRα^low/− hematovascular mesodermal precursors (HVMPs). HVMPs lack BL-CFC potential, but are highly enriched in cells that form hematoendothelial clusters on OP9. The endothelial stage of development was defined as expression of the typical endothelial markers VE-cadherin, CD31, and CD34 and the absence of the panhematopoietic marker CD43 (supplemental Table 1; see the Blood Web site).^,,,, Within the VE-cadherin⁺CD43⁻ population, HE cells (ie, cells lacking hematopoietic CFC potential but capable of forming blood cells after culture with stromal cells) were discriminated from non-HE cells based on lack of CD73 expression.^, The first hematopoietic progenitors emerging from the VE-cadherin⁺ population express CD235a, low levels of CD43, and lack CD41a expression. These cells have a unique potential to form hematopoietic colonies in the presence of FGF2 and hematopoietic cytokines, but also retain endothelial potential and therefore were designated as angiogenic hematopoietic progenitors (AHPs). Advanced hematopoietic development is associated with upregulation of CD43 expression; segregation of all hematopoietic CFCs to the CD43⁺ fraction^,,; and establishment of distinct subsets of CD43⁺ hematopoietic cells, including CD41a⁺CD235a⁺ erythro-megakaryocytic progenitors^,, and lin⁻CD34⁺CD43⁺CD45^+/− multipotent myelolymphoid progenitors.^,, Progressive acquisition of the angiogenic and hematopoietic program by differentiated cells is emphasized by green and red colors, respectively.

Figure 3

Schematic diagram demonstrating the opposite sequence of blood cell development between embryos and adults. In embryos, cells with restricted hematopoietic potential appear before HSC specification. In adults, hematopoiesis proceeds through gradual maturation of HSCs, leading to formation of progenitors with more restricted potential.

Figure 4

Potential approaches for de novo induction of HSCs.