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Chapter 9 Hematopoietic Stem Cell Biology 101

WNT-mediated maintenance of the HSC pool was demonstrated to BMP signaling is required for mesoderm formation and pattern-
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depend on intact NOTCH signaling, suggesting a deterministic ing, and BMPs are key regulators for the hematopoietic specification
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role for the NOTCH pathway in controlling the effects of WNT from mesoderm across different species (reviewed in ). BMP4
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signaling on the undifferentiated HSC pool. On the other hand, has been shown to modulate adult human HSC maintenance and
HSCs from mice engineered to conditionally express a stable form of proliferation in a concentration-dependent manner, with high BMP4
β-catenin were blocked in differentiation and failed to self-renew levels extending the survival of hematopoietic repopulating cells in ex
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leading to their exhaustion. 195,196 These results raise the possibility vivo cultures. However, in vivo, BMP signaling does not seem to be
that the prior report of HSC expansion in response to β-catenin required for adult HSC function as determined in mouse knockouts
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overexpression may have been affected by the use of Bcl2 transgenic for its signal transducers, SMAD1 and 5. On the other hand,
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mice. Alternatively, WNT signaling might have a more pronounced complete inhibition of the SMAD network has demonstrated the
role in vitro than in the more complex in vivo setting. 188 importance of SMAD proteins in regulating HSC self-renewal in vivo.
Some WNT ligands, e.g., WNT5A, are able to activate pathways Conditional deletion of Smad4 in mice led to a significantly reduced
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other than the canonical WNT pathway, depending on the particular ability of HSCs to repopulate primary and secondary recipients.
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WNT receptor context. In vivo activation of noncanonical WNT Also, retrovirus-mediated overexpression of the inhibitory Smad7
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signaling via systemic administration of WNT5A was shown to promoted HSC self-renewal in vivo. Taken together, these results
induce a greater than threefold increase in human CB HPC repopula- have been interpreted to indicate that SMAD4 positively regulates
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tion in NOD/SCID mice. More recent studies have demonstrated HSC self-renewal independently from its role as a mediator of SMAD
noncanonical WNT signaling in the BM niche to be required for pathway signaling. This hypothesis is supported by evidence demon-
HSC maintenance in vitro and in vivo. 198,199 Interestingly, it was strating that SMAD proteins can activate WNT signaling, 222,223 which
shown that Wnt5a expression is increased in ageing LT-HSCs. Induc- has been shown to promote HSC expansion as discussed earlier.
tion of Wnt5a in young mice induced ageing-associated HSC phe- As in other species and developmental contexts, 224,225,226 an intersec-
notypes, including apolarity, decreased repopulation capacity and tion between BMP4 and Hedgehog signaling has been described in
myeloid bias. Conversely, knocking down Wnt5a in old mice attenu- the human hematopoietic system. Hedgehog proteins play an essential
ated HSC ageing. 200 role in the embryonic development of a wide variety of organs, and,
Although activation of WNT signaling can induce HSC expan- like BMPs, they are required for mesoderm patterning. 227,228 Culture
sion, it is uncertain whether WNT signaling is indispensable for of human CB progenitor cells with Sonic Hedgehog (SHH), one of
normal hematopoiesis to occur. Conditional deletion of β-catenin in three human Hedgehog proteins, 229,230 promoted the expansion of
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adult BM progenitors did not impair their ability for multilineage cells capable of multilineage repopulation in NOD/SCID mice.
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reconstitution. In support of these results, hematopoietic cell abla- The addition of Noggin an endogenous inhibitor of BMP4, blocked
tion of porcupine, a membrane-bound O-acyl transferase essential for the effect of SHH on CB stem cell proliferation in vitro, whereas
WNT ligand secretion and receptor interaction, had no effect on Hedgehog inhibition did not block BMP4-induced hematopoietic
proliferation, differentiation, and self-renewal of adult HSCs in stem and progenitor cell (HSPC) proliferation, suggesting that BMP4
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vivo. Conversely, embryonic conditional knockout of β-catenin acts downstream of SHH in the regulation of human HSC growth.
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caused a deficiency in self-renewal of murine LT-HSCs, and HSCs Several other studies suggest that hedgehog signaling regulates HSC
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derived from Wnt3a mice failed to repopulate secondary recipi- growth. Mice heterozygous for the Hedgehog antagonist Patched1
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ents, suggesting that WNT signaling might have different roles in (Ptc1) were shown to have an expanded HSC compartment and
embryonic versus adult HSCs. their fetal HSCs exhibited increased colony-forming potential in
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serial plating assays. Interestingly, while conditional deletion of the
Hedgehog effector Smoothened (Smo) resulted in a profound loss of
TGF-β and Hedgehog Signaling LT-HSCs in the embryo, conditional deletion of Smo or pharma-
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cologic inhibition of Hedgehog had no effect on HSC content or
The TGF-β pathway represents a signaling mechanism that can be hematopoiesis in adult mice. 235,236 Thus, Hedgehog signaling during
activated by members of the TGF-β superfamily including TGF-β, embryogenesis might be required for certain aspects of HSC function
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activins and bone morphogenetic proteins (BMPs). Each of these important in adult life. 237
ligands binds to a specific receptor heterodimer composed of a type
I and II receptor, leading to the phosphorylation of a subset of
the receptor-regulated SMAD proteins (R-SMADs: SMAD1, 2, 3, CXCL12–CXCR4 Signaling
5, and 8). Thus activated R-SMADs then form a complex with
the common SMAD SMAD4, and translocate into the nucleus CXCL12 is a chemokine that is expressed by BM osteoblasts, ECs, and
to co-regulate target gene transcription. Another class of SMADs, perivascular stromal cells in the BM microenvironment and regulates
inhibitory SMADs (SMAD6 and 7) block TGF-β family signaling the homing and retention of HSCs. 238–240 Expression of CXCL12 or
by binding to R-SMADs. its receptor, CXCR4, is necessary for HSC maintenance in vivo. 239–241
TGF-β is one of the most potent inhibitors of HSC proliferation Recently, Ding et al reported that deletion of Cxcl12 from perivascular
in vitro, and neutralization of TGF-β releases HSCs from stromal cells or vascular ECs depleted HSCs in mice, whereas deple-
+
quiescence. 206–208 It has been suggested that TGF-β mediates cell cycle tion of Cxcl12 from nestin mesenchymal cells or osteoblasts had no
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inhibition in HSCs via upregulation of cyclin-dependent kinase effect on HSC numbers. Greenbaum et al also showed that deletion
inhibitors, p21, p27 and p57, as well as downregulation of cytokine of Cxcl12 in Prx1-expressing perivascular stromal cells led to HSC
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receptors. 209–213 The role of TGF-β in vivo appears to be more depletion, confirming the importance of CXCL12 signaling in the
complex. TGF-β likely functions as a negative hematopoietic regula- perivascular niche for HSC maintenance. A more detailed review of
tor in vivo as supported by the observation that deletion of TGF-β1 CXCL12-CXCR4 signaling in the context of the BM microenviron-
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results in extensive myelopoiesis in mice as well as defective homing ment, as well as a comprehensive review of HSC-HSC niche signaling
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of HSCs. Moreover, HSCs from mice with a conditional deletion interactions are presented in Chapter 11.
of the TGF-β type II receptor show increased cell cycling and
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impaired repopulation capacity. Conversely, TGF-β type I receptor
null mice display normal HSC self-renewal and regeneration in vivo, INTRINSIC PATHWAYS
although these HSCs exhibited increased proliferation in vitro. 217,218
The discrepancies between different knockout phenotypes could be Transcription Factors
caused by differences in expression levels of the TGF-β receptors in
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HSCs and thus different in vivo importance. Still, TGF-β is con- Transcription factors are proteins that bind specific sequences of DNA
sidered a critical signal for HSC quiescence also in vivo. 209 within promoter or enhancer regions to regulate the process of