IntroductionHematopoietic stem cells (HSCs) constitute a rare subpopulation in hematopoietic tissues and are uniquely defined by their ability to divide many times with full retention of their pluripotentiality. 1 This self-renewal function underlies the ability of HSCs to produce new blood cells throughout the life of the individual. It also allows them to regenerate the entire blood-forming system in vivo, including the HSC compartment, following their transplantation into irradiated recipients. 2,3 In mice, the ability of individual HSCs to permanently reconstitute all blood-cell lineages provides a functional end point for the specific quantification of HSCs in limiting-dilution transplantation assays. 4 The cells thus defined are operationally referred to as competitive repopulating units (CRUs), since these cells must compete with other HSCs whose presence is required for the survival of the host mice.Expansion of the HSC (CRU) population normally occurs in vivo during fetal and early adulthood. 5 Thereafter, the number of these cells remains stable with self-renewal divisions exactly balancing HSC losses by differentiation or death. However, a rapid and extensive net expansion of HSCs can be reinitiated in vivo following damage to the hematopoietic system. 6 Accumulating evidence points to the involvement of both extrinsic cues (eg, Steel factor, factors that activate gp130, 7 notch ligands, 8 and Wnt 9 ) as well as intrinsic regulators (eg, p21 cip1/waf1 , 10 Bmi-1, 11,12 and HoxB4 13-15 ) in controlling HSC self-renewal responses following their mitogenic activation in vitro or in vivo. Relatively little, however, is known about the signaling intermediates that direct or can modulate changes in HSC self-renewal when these cells are stimulated to proliferate in vivo.Signal transducer and activator of transcription 3 (STAT3) is an intermediate signaling molecule that allows ligand-induced signals from gp130 and the granulocyte colony-stimulating factor (G-CSF) receptor, and from various endogenous nonreceptor kinases such as Src, 16,17 to transactivate specific target genes. A key event in this process is the induced phosphorylation of the Tyr 705 residue in the Src-homology 2 (SH2) domain of STAT3, which then leads to the formation of STAT3 dimers and their translocation into the nucleus. Distinct biologic roles of STAT3 in various types of tissues including hematopoietic cells have been described in several studies. 18 In hematopoietic tissue, we showed that the forced expression of a dominant-negative (dn) form of Stat3 in fetal liver cells markedly reduced the competitive repopulating activity of the transduced HSCs in irradiated recipients of cotransplanted normal adult bone marrow cells. However, neither the proliferation nor the differentiation of slightly more mature progenitors in the original suspension of dnSTAT3-transduced fetal liver cells (detected in vivo as colony-forming units-spleen, CFU-Ss, or in vitro as colony-forming cells, CFCs) was affected. 19 Subsequently, it was found that induc...

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. STEM CELLS 2014;32:1313-1322

Low in vivo transduction efficiency and safety concerns have been hurdles for effective hematopoietic stem cell (HSC) gene therapy. Here, we investigate whether the safety and efficiency of retroviral gene transfer into HSCs can be improved by using human allogeneic umbilical cord blood (UCB)-derived supplements instead of fetal bovine serum (FBS). When CD34(+) cells were cultured ex vivo in UCB-derived serum (CBS) or plasma (CBP), comparable or higher maintenance of HSCs was observed than in FBS and serum-free substitution medium (SFM) as assessed by the frequency of positive engraftment and the level of engraftment in NOD/SCID mice after transplantation of cultured cells. CBS and CBP also exhibited higher level stabilization of retroviral particles than SFM during in vitro culture of retrovirus pseudotyped with gibbon ape leukemia virus or vesicular stomatitis virus glycoprotein. Retroviral gene transfer into CD34(+) cells performed with CBS or CBP resulted in increased gene transfer into CD34(+) cells and increased transduction of reconstituted bone marrow cells compared to transfers with SFM or FBS. The increased transduction of bone marrow cells was associated with a larger number of transduced progenitors in the recipient mice. Significant oligoclonality in the transduced progenitors, as determined by ligation-mediated polymerase chain reaction, suggested efficient retroviral targeting of multiple HSCs in the CBS- or CBP-supplemented media. Combined, our results show that allogeneic UCB-derived serum or plasma is a safe and easily accessible serum supplement that can support efficient retroviral gene transfer into HSCs for the clinical-grade manipulation of HSCs.

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