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The ability to manufacture human hematopoietic stem cells (HSCs) in the laboratory holds enormous promise for cellular therapy of human blood diseases. Several differentiation protocols have been developed to facilitate the emergence of HSCs from human pluripotent stem cells (PSCs). Most approaches employ a stepwise addition of cytokines and morphogens to recapitulate the natural developmental process. However, these protocols globally lack clinical relevance and uniformly induce PSCs to produce hematopoietic progenitors with embryonic features and limited engraftment and differentiation capabilities. This review examines how key intrinsic cues and extrinsic environmental inputs have been integrated within human PSC differentiation protocols to enhance the emergence of definitive hematopoiesis and how advances in genomics set the stage for imminent breakthroughs in this field. [ABSTRACT FROM AUTHOR]

Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA. [ABSTRACT FROM AUTHOR]

Homology-directed gene editing of hematopoietic stem and progenitor cells (HSPCs) is a promising strategy for the treatment of inherited blood disorders, obviating many of the limitations associated with viral vector-mediated gene therapies. The use of CRISPR/Cas9 or other programmable nucleases and improved methods of homology template delivery have enabled precise ex vivo gene editing. These transformative advances have also highlighted technical challenges to achieve high-efficiency gene editing in HSPCs for therapeutic applications. In this review, we discuss recent pre-clinical investigations utilizing homology-mediated gene editing in HSPCs and highlight various strategies to improve editing efficiency in these cells. [ABSTRACT FROM AUTHOR]

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity. [ABSTRACT FROM AUTHOR]