Engineering Hematopoietic Stem Cells: Lessons from Development

Abstract

Cell engineering has brought us tantalizingly close to the goal of deriving patient-specific hematopoietic stem cells (HSCs). While directed differentiation and transcription factor-mediated conversion strategies have generated progenitor cells with multilineage potential, to date, therapy-grade engineered HSCs remain elusive due to insufficient long-term self-renewal and inadequate differentiated progeny functionality. A cross-species approach involving zebrafish and mammalian systems offers complementary methodologies to improve understanding of native HSCs. Here, we discuss the role of conserved developmental timing processes in vertebrate hematopoiesis, highlighting how identification and manipulation of stage-specific factors that specify HSC developmental state must be harnessed to engineer HSCs for therapy.

Figure 1. Timing of hematopoietic maturation across species

The relative timing of hematopoiesis at specific anatomic sites in the human (blue), mouse (red), and zebrafish (green) are shown. Although the pace of hematopoietic maturation varies in each organism, hematopoiesis matures in highly conserved patterns through analogous organs prenatally and postnatally, with primitive hematopoiesis occurring in extraembryonic mesoderm derived cells.

Figure 2. LIN28 and let-7 in developmental timing and hematopoiesis

Maturation of definitive hematopoiesis from the fetal to adult stage is depicted. Hematopoiesis shifts from a fetal state characterized by rapid HSC self-renewal, fetal-specific lymphoid output, erythroid dominant myeloerythropoiesis, and, in humans, fetal globin expression, to the adult stage characterized by HSC quiescence, predominance of granulopoiesis, adult lymphopoiesis, and adult globin expression in humans. Lin28b is downregulated during maturation, allowing for increase in let-7 microRNAs. Effects of ectopic Lin28b/LIN28B expression on the timing of hematopoietic maturation are shown by arrows connecting adult and fetal hematopoietic populations. The broken arrow hypothesizes a possible effect of Lin28b modulation of granulopoiesis and erythropoiesis. Abbreviations: RBC, red blood cell; PMN, polymorphonuclear neutrophil; CMP, common myeloid progenitor; MEP, megakaryocyte erythroid progenitor; GMP, granulocyte macrophage progenitor; CLP, common lymphoid progenitor; MZ, marginal zone.

Figure 3. Examples of hematopoietic maturation in blood derivation strategies

This diagram highlights several key examples of blood derivation strategies showing the relative timing of developmental events, and classification of derived cells at stages of normal hematopoietic maturation. Examples of strategies using morphogen directed differentiation and conversion/respecification are depicted. The strategies employed by the Keller group typify morphogen directed differentiation approaches to hemogenic cell induction (Kennedy et al., 2012; Sturgeon et al., 2014). Sequential exposure of PSC EBs to morphogen cocktails initially specifies mesoderm fate, followed by hematopoietic fate induction by hematopoietic cytokines (Kennedy et al., 2012; Sturgeon et al., 2014). Though several studies have succeeded in reprogramming somatic cells to hematopoietic lineages (Pereira et al., 2013; Riddell et al., 2014), we present one example (Batta et al., 2014) whereby mouse fibroblasts were reprogrammed by expression of hematopoietic transcription factors, eventually producing cells with short-term engraftment in vivo. PSC reprogramming is an approach also used by multiple groups to generate hematopoietic cells (Doulatov et al., 2013; Elcheva et al., 2014). Here, we summarize the strategy used by Doulatov and colleagues, where hematopoietic progenitors generated by morphogen directed differentiation were respecified to engraftable definitive progenitors, and lineage output analyzed in vivo (Doulatov et al., 2013). Finally, two distinct approaches leveraging understanding of EHT are shown (Pereira et al., 2016; Sandler et al., 2014). Sandler and colleagues reprogrammed human endothelial cells with transcription factors that promoted an EHT-like process, generating multilineage engraftable MPP-like cells (Sandler et al., 2014). Pereira and colleages isolated mouse placental hemogenic precursors based on an immunophenotype identified on endothelia-like cells generated in their prior somatic cell reprogramming (Pereira et al., 2013), showing that these cells could be induced to engraft and undergo multilineage hematopoiesis (Pereira et al., 2016). Known stage-specific hematopoietic regulators are shown in bold. Abbreviations: hPSC, human pluripotent stem cell; EBs, embryoid bodies.