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Chapter 9 Hematopoietic Stem Cell Biology 107

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of human HSCs in culture. Jaroscak et al tested the combination progenitors with short-term myeloid and erythroid engraftment
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of FLT3 ligand, a GM-CSF/IL-3 fusion protein, and erythropoietin potential. This and similar approaches are characterized by the lack
in a continuous perfusion culture system as a means to expand human of robust lymphoid potential, likely because these progenitors are
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CB cells before transplant. Similarly, Shpall et al tested the capacity developmentally still too immature. 484
of SCF, granulocyte colony-stimulating factor (G-CSF), and mega- In light of the obstacles in generating HSCs from ESCs or iPSCs,
karyocyte growth and differentiation factor to expand human CB approaches to directly reprogram somatic cells into HSCs, or trans-
cells that were then transplanted into adult CB transplant recipients. differentiate them to hematopoietic cells are being explored as
An alternative approach to cytokine-based expansion of human CB alternative strategies. The concept of direct reprogramming was first
cells was suggested by Peled et al 464–466 who demonstrated a 159-fold demonstrated with the conversion of embryonic fibroblasts into
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increase in human CD34 cells after seven-week culture with a copper contracting myocytes by just the transcription factor MyoD.
chelator, tetraethylenepentamine (TEPA), and cytokines. Subse- Pluripotency-related factors are upregulated during endogenous
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quently, de Lima et al reported the safety and feasibility of culturing reconstitution of mouse hematopoiesis after irradiation. Thus,
human CB cells with TEPA and SCF, FLT3 ligand, IL-6, and throm- studies successfully reprogramming human skin fibroblasts directly
bopoietin followed by transplantation into patients in a phase I/II into HSPC-like cells used the pluripotency factors OCT4 or SOX2
clinical trial. Although each of these clinical trials has shown the together with a specific cytokine cocktail. 486,487 While these cells
feasibility of transplanting ex vivo–cultured CB cells, none demon- engrafted, they again lacked lymphoid potential. In contrast to these
strated substantial acceleration in hematopoietic cell engraftment in examples of indirect lineage conversion via a less differentiated state,
CB transplant recipients compared to historical controls. However, direct lineage conversion (or transdifferentiation) attempts have
the TEPA plus cytokine strategy is being tested further in a phase II/ included the enforced expression of transcription factors critical for
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III study in several countries, including the United States. 166 normal hematopoiesis. In analogy to the HSC-producing hemo-
Several other clinical trials have recently indicated progress toward genic endothelium, human umbilical-vein ECs cultured on an artifi-
the clinical expansion of human CB HSCs for therapeutic purposes. cial vascular niche and overexpressing four transcription factors
De Lima et al reported a median time to neutrophil engraftment of (FOSB, GFI1, RUNX1 and PU.1) yielded serially transplantable
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15 days in recipients of one unmanipulated CB unit plus CB cells hematopoietic colonies. However, these cells did not differentiate
cultured with mesenchymal stromal cells, compared to 24 days in into T cells. In another approach demonstrating the benefit of the
historical controls, although long-term donor hematopoiesis derived niche, lymphoid or myeloid progenitors were transduced with a
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almost exclusively from the unmanipulated cord blood unit. Lastly, transcription factor cocktail (HLF, LMO2, PBX1, PRDM5,
Horwitz et al reported that transplantation of one unmanipulated RUNX1T1 and ZFP37) and matured in irradiated mice to yield
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CB unit and the progeny of 21-day culture of human CB cells with serially transplantable HSCs producing all lineages. Despite this
nicotinamide produced earlier neutrophil recovery (13 versus 25 progress in mice, fully functional human HSCs have not yet been
days for historical controls) and dominant engraftment from the generated in vitro. In addition, future studies will have to focus on
nicotinamide-treated CB unit in eight of 11 treated patients. 469 strategies to avoid the risk of malignant transformation inherent in
any directed differentiation or cellular reprogramming method. 491
GENERATING HEMATOPOIETIC STEM CELLS FROM HEMATOPOIETIC STEM CELL REGENERATION
PLURIPOTENT STEM CELLS AND BY REPROGRAMMING
OF SOMATIC CELLS Although much is now known about the intrinsic and extrinsic
mechanisms that regulate adult HSC self-renewal and differentia-
Globally more than 50,000 patients per year receive allogeneic and tion, 1,166,188 the process through which HSCs regenerate after injury
autologous HSC transplantations as treatments for congenital and (e.g., chemotherapy or radiation) remains less well understood. Suc-
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acquired hematopoietic diseases and other malignancies. At present, cessful delineation of the mechanisms that control HSC regeneration
the only cell sources for HSC transplantations are BM, CB, or has significant therapeutic potential because a large proportion of
mobilized peripheral blood. However, insufficient numbers, shelf-life patients with cancer receive myelosuppressive or myeloablative
concerns as well as immunologic incompatibility leading to graft- therapy during the course of their disease. Signaling through the
versus-host disease, even in human leukocyte antigen-matched grafts, BMP and WNT signaling pathways has been shown to be necessary
limits their availability. 471,472 One option to generate more HSCs is for hematopoietic regeneration to occur in zebrafish after sublethal
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by expanding existing HSCs in vitro as described earlier. Despite irradiation. These authors further demonstrated that SMAD and
substantial efforts, this has proven difficult; because of their tendency TCF, the downstream effectors of BMP and WNT signaling, respec-
to differentiate in culture, the expansion of HSCs is not very efficient tively, couple with master regulators of myeloid and erythroid
and does often not lead to fully functional HSCs in terms of their differentiation (C/EBPα and GATA1) to drive lineage-specific
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migratory behavior and long-term multilineage reconstitution regeneration. In a murine model of hematopoietic injury, Congdon
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potential. 461,473,474 et al showed that Wnt10b expression is increased in BM stromal
The observation in 1981 that embryonic stem cells (ESCs) could cells in response to irradiation, and WNT signaling is activated in
be derived from mouse or, later, human blastocysts 475–477 fueled BM HSCs after irradiation. As discussed earlier, in a zebrafish model,
experiments to differentiate HSCs from ESCs. In 2006, the discovery activation of WNT signaling during hematopoietic regeneration is
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that mouse or human fibroblasts could be reprogrammed to induced modulated by PGE 2. WNT reporter activity was responsive to
pluripotent stem cells (iPSCs) by retroviral transduction with the PGE 2 treatment, and the effect of Wnt8 toward enhancing hemato-
same four factors, OCT3/4, SOX2, KLF4, and c-Myc, 478,479 opened poietic recovery after sublethal irradiation was inhibited by adminis-
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the door for the possibility of autologous stem-cell based therapies in tration of indomethacin, a PGE 2 antagonist. NOTCH signaling
the clinic. 480,481 Since its first report this technology has been con- has also been implicated in the regulation of hematopoietic regenera-
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stantly modified and is allowing the generation of iPSC lines from tion after stem cell transplantation. Deletion of Notch2, but not
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patients with a variety of blood disorders (references in ). This Notch1, was shown to delay myeloid reconstitution in mice after stem
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approach presents a new opportunity for disease modeling and drug cell transplantation. These data suggest that the BMP, WNT, and
screening. NOTCH pathways are attractive mechanistic targets for strategies to
Numerous methods for directed differentiation of HSCs from augment hematopoietic regeneration after myelosuppressive therapy.
ESCs or more recently iPSCs have been developed, but so far none Additional signaling pathways have been implicated in regulat-
yield long-lived cells with full HSC functionality. In a recent study, ing hematopoietic regeneration. Deletion of plasminogen (Plg), a
inducible expression of five transcription factors, HOXA9, RORA, fibrinolytic factor, was shown to prevent HPC proliferation and
ERG, SOX4, and MYB imparted human ESC- and iPSC-derived recovery after 5-fluorouracil (5-FU)-induced myelosuppression in