Molecular integration of HoxB4 and STAT3 for self-renewal of hematopoietic stem cells: a model of molecular convergence for stemness.

The upregulation of HoxB4 promotes self-renewal of hematopoietic stem cells (HSCs) without overriding the normal stem cell pool size. A similar enhancement of HSC self-renewal occurs when signal transducer and activator of transcription 3 (STAT3) is activated in HSCs. In this study, to gain insight into the functional organization of individual transcription factors (TFs) that have similar effects on HSCs, we investigated the molecular interplay between HoxB4 and STAT3 in the regulation of HSC self-renewal. We found that while STAT3-C or HoxB4 similarly enhanced the in vitro self-renewal and in vivo repopulating activities of HSCs, simultaneous transduction of both TFs did not have additive effects, indicating their functional redundancy in HSCs. In addition, activation of STAT3 did not cause changes in the expression levels of HoxB4. In contrast, the inhibition of STAT3 activity in HoxB4-overexpressing hematopoietic cells significantly abrogated the enhancing effects of HoxB4, and the upregulation of HoxB4 caused a ligand-independent Tyr-phosphorylation of STAT3. Microarray analysis revealed a significant overlap of the transcriptomes regulated by STAT3 and HoxB4 in undifferentiated hematopoietic cells. Moreover, a gene set enrichment analysis showed significant overlap in the candidate TFs that can recapitulate the transcriptional changes induced by HoxB4 or STAT3. Interestingly, among these common TFs were the pluripotency-related genes Oct-4 and Nanog. These results indicate that tissue-specific TFs regulating HSC self-renewal are functionally organized to play an equivalent role in transcription and provide insights into the functional convergence of multiple entries of TFs toward a conserved transcription program for the stem cell state.

Microenvironmental remodeling as a parameter and prognostic factor of heterogeneous leukemogenesis in acute myelogenous leukemia.

Acute myelogenous leukemia (AML) is a heterogeneous disorder characterized by clonal proliferation of stem cell-like blasts in bone marrow (BM); however, their unique cellular interaction within the BM microenvironment and its functional significance remain unclear. Here, we assessed the BM microenvironment of AML patients and demonstrate that the leukemia stem cells induce a change in the transcriptional programming of the normal mesenchymal stromal cells (MSC). The modified leukemic niche alters the expressions of cross-talk molecules (i.e., CXCL12 and JAG1) in MSCs to provide a distinct cross-talk between normal and leukemia cells, selectively suppressing normal primitive hematopoietic cells while supporting leukemogenesis and chemoresistance. Of note, AML patients exhibited distinct heterogeneity in the alteration of mesenchymal stroma in BM. The distinct pattern of stromal changes in leukemic BM at initial diagnosis was associated with a heterogeneous posttreatment clinical course with respect to the maintenance of complete remission for 5 to 8 years and early or late relapse. Thus, remodeling of mesenchymal niche by leukemia cells is an intrinsic self-reinforcing process of leukemogenesis that can be a parameter for the heterogeneity in the clinical course of leukemia and hence serve as a potential prognostic factor.

Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state.

With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1α (Hif-1α), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1α stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1α in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1α during such long-term biological processes. Using this model, we show that the stabilization of Hif-1α proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1α stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1α proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.

Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγ(null) mice.

Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ(null) (NSG) mice. By isolating fucosylated and nonfucosylated CD34(+) cells from CB, we showed that only fucosylated CD34(+) cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34(+) cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34(+) cells, we hypothesized that increasing the proportion of CD34(+) cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34(+) fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.

Stochastic acquisition of a stem cell-like state and drug tolerance in leukemia cells stressed by radiation.

A rare population of leukemia cells have the properties of leukemia stem cells (LSCs) and cause resistance to therapy, but their development is not clearly understood. In the current study, we show that a higher resistance to cytotoxic drug (Ara-C) can be developed in the subpopulation of promyelocytic leukemia cells that survived radiation treatment. These drug-tolerant leukemia cells (DTLs) are not observed immediately after radiation despite extensive genetic instability in the cells, but appear in 3 weeks of recovery culture. Moreover, when the single cell-derived clones were examined by clonal trafficking, no correlation between radio-resistant and chemo-resistant leukemic clones was detected, indicating that the resistance is developed by active acquisition of the resistance without clonal predisposition. Interestingly, the DTLs mimicked the characteristics of LSCs exhibiting leukemia-initiating activities and lower levels of reactive oxygen species or a higher level expression of bmi-1 as well as higher resistance to retinoic acid-induced differentiation compared to parental leukemic cells. These studies show that an active reacquisition of stem cell-like properties can occur in the leukemia cells to develop resistance to treatments and that such reacquisition process of leukemic cells occurs in a stochastic manner triggered by radiation stress on leukemic cells.

Intramarrow injection of beta-catenin-activated, but not naive mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow.

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naïve MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with beta-catenin-activated MSCs. These results reveal that naive MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/beta-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.

Early apoptosis in CD34+ cells as a potential heterogeneity in quality of cryopreserved umbilical cord blood.

The increased use of umbilical cord blood (UCB) raises issues regarding the quality of cryopreserved UCB. This study investigated whether early apoptosis of CD34+ cells is part of the functional heterogeneity of cryopreserved UCB. Annexin V binding of CD34+ PI(-) cells showed wide variations in both fresh and cryopreserved UCBs, with greater variation among units frozen for > 5 years. Xenotransplantation of sorted cells into non-obese diabetic severe combined immunodeficient mice demonstrated that the Annexin V assay identified most repopulating activities in UCB units. Thus, early apoptosis of CD34+ cells could influence the outcome of transplantation using cryopreserved UCB.