Page 129 - Hematology_ Basic Principles and Practice ( PDFDrive )

100 Part II Cellular Basis of Hematology

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is unknown but presumably relates to the ex vivo handling of cells HSC cultures. Furthermore, Jagged2-mediated activation of
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for transplantation or the stress of increased proliferation following NOTCH signaling inhibits the differentiation of human CB CD34
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transplantation. Furthermore, while it is commonly thought that in cells, and culture of human CB HSCs with soluble human Jagged1
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myeloid malignancies the “cell of origin”, which acquires the first induces HSC expansion ex vivo. NOTCH ligands that are expressed
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cancer-promoting mutation is an HSC because of their extended in the surrounding BM niche are thought to be critical in promoting
lifespan, these recent studies suggest that long-lived progenitors HSC maintenance through the activation of NOTCH receptors
might be equally suitable candidates for tumor-initiating cells. 160 expressed on HSCs. For example, BM osteoblasts express Jagged1 and
blocking NOTCH activation with a γ-secretase inhibitor significantly
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decreases murine HSC expansion in BM osteoblast cocultures.
REGULATION OF HEMATOPOIETIC STEM CELL FATE Similarly, sinusoidal ECs that express NOTCH ligands stimulate
−/−
−/−
expansion of wild-type but not Notch1 Notch2 LT-HSCs in
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HSCs have the capacity to generate more stem cells, a process called vitro. However, although deletion of Jagged1, Notch1, or Rbpj
self-renewal, and to produce cells that differentiate into the entire results in impaired embryonic hematopoiesis in the mouse, the physi-
spectrum of mature hematopoietic cells. The balance between these ologic role of NOTCH signaling in the maintenance of the adult
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fate choices is thought to be regulated by the type of cell division that HSC pool in vivo is controversial. Deletion of Jagged1 was shown
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HSCs undergo. Asymmetrical cell divisions result in one HSC (self- to have no effect on HSC content in mice, and deletion of Rbpj
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renewal) and one committed HPC, allowing the maintenance of the caused no defect in HSC repopulating capacity. Interestingly, while
stem cell pool while concomitantly ensuring the supply of differenti- neither Notch1 nor Notch2 were found to be required for HSC
ated cells. However, during development and regeneration the stem cell function under homeostatic conditions in vivo, 170,177 challenging the
pool must have the capacity to expand. This can be achieved by sym- BM with chemotherapy or radiation in the presence of a conditional
metrical cell divisions, which will lead to two daughter HSCs capable Notch2 deletion resulted in more rapid myeloid differentiation at the
of self-renewing. Another outcome of symmetric division would be two expense of HSC self-renewal. 170
HPCs, diminishing and ultimately exhausting the HSC pool. The fate In keeping with the evidence obtained from mouse studies that
decision governing this balance between self-renewal and differentia- activation of NOTCH signaling can induce HSC expansion, Delaney
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tion is regulated by cell-intrinsic including transcriptional and epigen- et al showed that serum-free culture of human CB progenitor
etic mechanisms that are intertwined with cell-extrinsic mechanisms cells with immobilized Delta1 plus cytokines for three weeks yielded
from the microenvironment or by the action of systemic factors. a 5.3-fold increase in human hematopoietic cell engraftment in
transplanted NOD/SCID mice. This group subsequently completed
a phase I clinical trial showing that transplantation of CB cells,
Extrinsic Regulation which had been expanded with immobilized Delta1 along with an
unmanipulated CB unit shortened the time interval to neutrophil
The past three decades have yielded substantial progress in the dis- recovery (median, 16 days) compared with a cohort that received
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covery and characterization of intrinsic and extrinsic mechanisms that two unmanipulated CB units (median, 26 days). Of note, in this
regulate HSC self-renewal and differentiation. Despite this, the phase I study, the unmanipulated CB cells demonstrated dominant
translation of these discoveries into the development of translatable engraftment by day 80 after transplant and in seven of eight reported
methods to expand human HSCs ex vivo or therapeutics to induce recipients, ex vivo expanded CB cells were not detectable in recipients
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HSC expansion in vivo has proven to be difficult. Therefore, further by day 40 posttransplant. The extinction of the Delta1-expanded
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dissection of the mechanisms governing HSC function continues to CB units might be explained by T-cell depletion as donor CB CD8
be a high priority. The following pathways are extrinsically controlled T cells of a successful graft have been shown to mediate the rejection
and reflect unique mechanistic targets for the development of thera- of the other CB unit in the setting of double CB transplantation. 181
peutics to amplify the human HSC pool.
WNT Signaling
NOTCH Signaling
WNT signaling is initiated by the interaction of WNT ligands with
The NOTCH signaling pathway has been shown to have an impor- the so-called Frizzled/LRP (lipoprotein receptor-related protein) cell
tant role in regulating the development of the central nervous system, surface receptor complex. In the absence of ligand binding, the WNT
eye, muscle, hematopoietic system, and germline, among others. 163,164 signal transducer β-catenin is phosphorylated by kinases such as
To date, four NOTCH receptors have been identified (NOTCH1–4) glycogen synthase kinase-3 beta (GSK-3β), leading to its rapid deg-
as well as five ligands for Notch receptors (JAGGED1 and 2 and radation. Upon activation of the canonical WNT pathway, β-catenin
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DELTA1, 3, and 4). Ligand receptor interaction on HSCs induces is no longer phosphorylated and thereby stabilized, translocates into
two NOTCH cleavage steps, the last of which is mediated by γ-secretase the nucleus and interacts with transcription factors of the T-cell
and releases the constitutively active NOTCH-intracellular domain factor/lymphoid enhancer factor (LEF/TCF) family to regulate
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(NICD). The NICD then translocates to the nucleus, interacts with expression of target genes. Several lines of evidence implicate WNT
the DNA-binding protein RBPJ (recombination signal binding signaling in the regulation of HSC self-renewal and differentiation.
protein for immunoglobulin kappa J region) and initiates transcrip- First, WNT proteins have been shown to be expressed at sites of
tion of target genes such as the transcription factors HES1 and embryonic and fetal hematopoiesis, and WNT ligands, receptors and
HES5 (mammalian homologues of Drosophila hairy and Enhancer LEF/TCF transcription factors are expressed by adult HSCs as well
of split). 166–169 Depending on the specific NOTCH ligand, different as the BM microenvironment. 183,184 Second, ample evidence suggests
NOTCH receptors and thus different NOTCH target genes are acti- that activation of WNT signaling is capable of promoting HSC
vated, leading to diverse cellular outcomes. For example, NOTCH1 expansion, at least in vitro. 185–189 Using mice transgenic for human
activation by Delta1 and 4 is required for T-cell differentiation while BCL2 enabling HSCs to survive in the presence of stem cell factor
it inhibits differentiation of the B-cell lineage. Jagged2- or Delta1- (SCF) alone, 190,191 it was shown that BM KTLS cells treated with
mediated activation of NOTCH2 inhibits myeloid differentiation purified WNT3A protein or transduced with active β-catenin ex vivo
and induces the generation of LT-HSCs and MPPs. 167,170 resulted in their expansion and multilineage reconstitution of com-
Activation of NOTCH signaling is sufficient to induce ex vivo petitively transplanted recipients. 185,189 In a related study, treating
HSC expansion. Retroviral expression of the Notch1 ICD in murine immune-deficient recipient mice with a GSK-3β inhibitor after
HSCs leads to the generation of an immortal, cytokine-dependent transplantation of lineage-depleted human CB HSCs increased their
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cell line with multilineage in vivo repopulating capacity, and engraftment and repopulating capacity, which was accompanied by
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immobilized Delta1 promotes a several-log expansion of murine faster recovery from posttransplant cytopenia. Importantly,