OP9 stroma augments survival of hematopoietic precursors and progenitors during hematopoietic differentiation from human embryonic stem cells

The cellular mechanism and target cell affected by stromal microenvironments in augmenting hematopoietic specification from pluripotent human embryonic stem cells (hESCs) has yet to be evaluated. Here, in contrast to aorta-gonad-mesonephros-derived S62 stromal cells, OP9 cells inhibit apoptosis and also augment the proliferation of hemogenic precursors prospectively isolated from human embryoid bodies. In addition, OP9 stroma supported cells within the primitive hematopoietic compartment by inhibiting apoptosis of CD45(+)CD34(+) cells committed to the hematopoietic lineage, but have no effect on more mature blood (CD45(+)CD34(-)) cells. Inability of hESC-derived hematopoietic cells cocultured with OP9 stromal cells to engraft in both the adult and newborn NOD/SCID mice after intrafemoral and intrahepatic injection illustrated that although OP9 stromal cells augment hESC-derived hematopoiesis and progenitor output, this optimized environment does not confer or augment repopulating function of specified hematopoietic cells derived from hESCs. OP9 coculture also increases hematopoietic progenitors output from hemogenic precursors overexpressing HOXB4. Our study demonstrates that OP9 cells support both hemogenic precursors and their primitive hematopoietic progeny, thereby providing the first evidence toward understanding the cellular targets and mechanisms underlying the capacity of OP9 stromal cells to support hematopoiesis from ESCs and define the future steps required to achieve the global goal of generating bona fide human hematopoietic stem cells from ESC lines. Disclosure of potential conflicts of interest is found at the end of this article.     

The HOXB4 homeoprotein differentially promotes ex vivo expansion of early human lymphoid progenitors

The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34(+) hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.     

Effects of HOXB4 overexpression on ex vivo expansion and immortalization of hematopoietic cells from different species

Overexpression of the human HOXB4 has been shown to induce the expansion and self-renewal of murine hematopoietic stem cells. In preparation for clinical studies, we wished to investigate the effects of HOXB4 on cells from other species, in particular preclinical large animals such as dogs and nonhuman primates. Thus, we transduced CD34(+) cells from nonhuman primates, dogs, and humans with a HOXB4-expressing gammaretroviral vector and a yellow fluorescent protein-expressing control vector. Compared with the control vector, HOXB4 overexpression resulted in a much larger increase in colony-forming cells in dog cells (28-fold) compared with human peripheral blood, human cord blood, and baboon cells (two-, four-, and fivefold, respectively). Furthermore, we found that HOXB4 overexpression resulted in immortalization with sustained growth (>12 months) of primitive hematopoietic cells from mice and dogs but not from monkeys and humans. This difference correlated with increased levels of retrovirally overexpressed HOXB4 in dog and mouse cells compared with human and nonhuman primate cells. The immortalized cells did not show any evidence of insertional mutagenesis or chromosomal abnormalities. Competitive congenic transplantation experiments showed that HOXB4-expanded mouse cells engrafted well after 1 or 3 months of expansion, and no leukemia was observed in mice. Our findings suggest that the growth promoting effects of HOXB4 are critically dependent on HOXB4 expression levels and that this can result in important species-specific differences in potency. Disclosure of potential conflicts of interest is found at the end of this article.     

Pre-differentiated human committed T-lymphoid progenitors promote peripheral T-cell re-constitution after stem cell transplantation in immunodeficient mice

T-cell re-constitution after allogeneic stem cell transplantation (alloSCT) is often dampened by the slow differentiation of human peripheral blood CD34(+) (huCD34(+) ) hematopoietic stem cells (HSCs) into mature T cells. This process may be accelerated by the co-transfer of in vitro-pre-differentiated committed T/NK-lymphoid progenitors (CTLPs). Here, we analysed the developmental potential of huCD34(+) HSCs compared with CTLPs from a third-party donor in a murine NOD-scid IL2Rγ(null) model of humanised chimeric haematopoiesis. CTLPs (CD34(+) lin(-) CD45RA(+) CD7(+) ) could be generated in vitro within 10 days upon co-culture of huCD34(+) or cord blood CD34(+) (CB-CD34) HSCs on murine OP9/N-DLL-1 stroma cells but not in a novel 3-D cell-culture matrix with DLL-1(low) human stroma cells. In both in vitro systems, huCD34(+) and CB-CD34(+) HSCs did not give rise to mature T cells. Upon transfer into 6-wk-old immune-deficient mice, CTLPs alone did not engraft. However, transplantation of CTLPs together with huCD34(+) HSCs resulted in rapid T-cell engraftment in spleen, bone marrow and thymus at day 28. Strikingly, at this early time point mature T cells originated exclusively from CTLPs, whereas descendants of huCD34(+) HSCs still expressed a T-cell-precursor phenotype (CD7(+) CD5(+) CD1a(+/-) ). This strategy to enhance early T-cell re-constitution with ex vivo-pre-differentiated T-lymphoid progenitors could bridge the gap until full T-cell recovery in severely immunocompromised patients after allogeneic stem cell transplantation.     

Sustained long-term engraftment and transgene expression of peripheral blood CD34+ cells transduced with third-generation lentiviral vectors

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.     

Gene expression analysis of hematopoietic progenitor cells identifies Dlg7 as a potential stem cell gene

Inducible hematopoietic stem/progenitor cell lines represent a model for studying genes involved in self-renewal and differentiation. Here, gene expression was studied in the inducible human CD34+ acute myelogenous leukemia cell line KG1 using oligonucleotide arrays and suppression subtractive cloning. Using this approach, we identified Dlg7, the homolog of the Drosophila Dlg1 tumor suppressor gene, as downregulated at the early stages of KG1 differentiation. Similarly, Dlg7 was expressed in normal purified umbilical cord blood CD34+CD38- progenitors but not in the more committed CD34+CD38+ population. Dlg7 expression was not detected in differentiated cells obtained from hematopoietic colonies, nor was expression detected in purified T-cells, B-cells, and monocytes. When analyzed in different types of stem cells, Dlg7 expression was detected in purified human bone marrow-derived CD133+ progenitor cells, human mesenchymal stem cells, and mouse embryonic stem (ES) cells. Overexpression of Dlg7 in mouse ES cells increased their growth rate and reduced the number of EBs emerging upon differentiation. In addition, the EBs were significantly smaller, indicating an inhibition in differentiation. This inhibition was further supported by higher expression of Bmp4, Oct4, Rex1, and Nanog in EBs overexpressing Dlg7 and lower expression of Brachyury. Finally, the Dlg7 protein was detected in liver and colon carcinoma tumors but not in normal adjacent tissues, suggesting a role for the gene in carcinogenesis. In conclusion, our results suggest that Dlg7 has a role in stem cell survival, in maintaining stem cell properties, and in carcinogenesis. Disclosure of potential conflicts of interest is found at the end of this article.     

Notch-induced hIL-6 production facilitates the maintenance of self-renewal of hCD34+ cord blood cells through the activation of Jak-PI3K-STAT3 pathway

Ex vivo expansion of CD34(+) stem cells in contact culture between hCD34(+)CD38(-)Lin(-) cord blood stem cells and human delta-like-expressing AFT024 feeder cells revealed increased amounts of stemness-related proteins such as HoxB4, GATA2, Bmi-1, and p21 and anti-apoptotic proteins such as Bcl-2, Bcl-xL, Mcl-1, and phospho-Bad, when compared with control or noncontact culture. Production of human IL-6 (hIL-6) was markedly elevated in the culture, but was profoundly inhibited by treatment with γ-secretase inhibitor. In addition, Notch-induced activation of STAT3 was directly involved in gene expression of hIL-6 and soluble hIL-6Rα, indicating the close linkage between Notch signaling and hIL-6 production. Furthermore, depletion of soluble hIL-6 (with hIL-6-specific antibodies) and inhibition of IL-6-mediated signals (with a Jak1 inhibitor and wortmannin) severely affected the maintenance of self-renewal of hCD34(+) cord blood cells. It was also observed that the ex vivo expanded CD34(+) cord blood cells were induced to reconstitute human immune cells in nonobese diabetic mice with severe combined immunodeficiency when compared with freshly isolated CD34(+) cord blood cells. Together, these results strongly demonstrate that Notch signaling in the "cell-to-cell contact" between hCD34(+) cord blood and delta-like-expressing AFT024 feeder cells facilitates maintenance of self-renewal of hCD34(+) cord blood cells through direct regulation of hIL-6 production.     

Cytokine expansion culture of cord blood CD34+ cells induces marked and sustained changes in adhesion receptor and CXCR4 expressions

Recent studies have demonstrated defective bone marrow homing of hematopoietic stem cells after cytokine expansion culture. Adhesion receptors (ARs) are essential to the homing process, and it is possible that cytokine culture modulates AR expression. We studied changes in expression of very late antigen-4 (VLA-4), VLA-5, L-selectin, leukocyte function-associated antigen-1 (LFA-1), CD44, and the stromal cell-derived factor-1 (SDF-1) receptor, CXCR4, during cytokine culture of cord blood (CB) CD34(+) cells. Expression of ARs was studied by flow cytometry on CB CD34(+) cells in whole blood, after purification and during culture for up to 10 days. Cells were cultured with stem cell factor (SCF), thrombopoietin (TPO), Flt3-ligand (Flt3), and G-CSF. Results showed that 80% or more of uncultured CD34(+) cells were positive for VLA-4, L-selectin, LFA-1, CD44, and CXCR4 while 50% were positive for VLA-5. Purification of CD34(+) cells did not affect AR expression, but cytokines increased expression three- to nine-fold throughout the 10-day culture period. In contrast, expression of CXCR4 decreased. Expression changes of ARs and CXCR4 on CD34(+)/CD38(-) cells mirrored those of the total CD34(+) population. The results indicate that cytokine culture significantly increases AR expression on CB CD34(+) cells, which may be related to the decrease in homing of cytokine-cultured hematopoietic stem cells.     

Highly efficient ex vivo expansion of human hematopoietic stem cells using Delta1-Fc chimeric protein

Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, interleukin (IL)-3, and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/gammac(null) mice, which showed myeloid, B, T, and natural killer cells as well as CD133(+)CD34(+) cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.     

Mitochondrial DNA sequence heterogeneity of single CD34+ cells after nonmyeloablative allogeneic stem cell transplantation

We applied a single-cell method to detect mitochondrial DNA (mtDNA) mutations to evaluate the reconstitution of hematopoietic stem cells (HSCs) and committed progenitor cells after nonmyeloablative allogeneic stem cell transplantation in humans. In a total of 1,958 single CD34(+) cells from six human leukocyte antigen-matched sibling donor and recipient pairs, individual CD34(+) clones were recognized based on the observed donor- or recipient-specific mtDNA sequence somatic alteration. There was no overall reduction of mtDNA heterogeneity among CD34(+) cells from the recipient after transplantation. Samples collected from two donors over time showed the persistence of certain CD34(+) clones marked by specific mutations. Our results demonstrate the feasibility of distinguishing donor and recipient individual CD34(+) clones based on mtDNA mutations during engraftment. HSCs were not limited in number, and similar mtDNA heterogeneity levels suggested representation of the total stem cell compartment during rapid hematopoietic reconstitution in the recipient. Disclosure of potential conflicts of interest is found at the end of this article.     

The competitive nature of HOXB4-transduced HSC is limited by PBX1: the generation of ultra-competitive stem cells retaining full differentiation potential

We previously showed that HOXB4 is a potent stimulator of hematopoietic stem cell (HSC) proliferation in vivo and ex vivo. As a result, HOXB4 overexpressing HSCs are 20- to 50-times more competitive than untransduced cells when transplanted into mice. By knocking down the expression of PBX1 (PBX1(K.D.)) in HOXB4 overexpressing cells, we now present the possibility of generating HSCs that are >20-times more competitive than those that overexpress HOXB4. The differentiation activity of these cells appears intact, since they competitively contributed to the reconstitution of normal myeloid and lymphoid compartments in vivo. We also show that the in vivo expansion of HOXB4-PBX1(K.D.)-expressing HSCs regenerated normal stem cell pools and did not lead to HSC levels above those detected in unmanipulated mice. The vigorous competitive nature of these cells in vivo compared to HOXB4-transduced HSCs suggests the existence of a distinct, non-cell autonomous mechanism that limits the expansion of HOXB4-transduced hemopoietic stem cells in mice.     

Promoting effects of serotonin on hematopoiesis: ex vivo expansion of cord blood CD34+ stem/progenitor cells, proliferation of bone marrow stromal cells, and antiapoptosis

Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5-hydroxytryptamine (5-HT)2 receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34+ cells, bone marrow (BM) stromal cell colony-forming unit-fibroblast (CFU-F) formation, and antiapoptosis of megakaryoblastic M-07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34+ cells to early stem/progenitors (CD34+ cells, colony-forming unit-mixed [CFU-GEMM]) and multilineage committed progenitors (burst-forming unit/colony-forming unit-erythroid [BFU/CFU-E], colony-forming unit-granulocyte macrophage, colony-forming unit-megakaryocyte, CD61+ CD41+ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45+, CD33+, CD14+ cells, BFU/CFU-E, and CFU-GEMM engraftment in BM of animals 6 weeks post-transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor stimulated BM CFU-F formation. In M-07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase-3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5-HT2 receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation. Disclosure of potential conflicts of interest is found at the end of this article.     

Mesenchymal stem cells secreting angiopoietin-like-5 support efficient expansion of human hematopoietic stem cells without compromising their repopulating potential

Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34(+) CD133(+) cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ~60-fold by limiting dilution assay in NOD-scid Il2rg(-/-) (NSG) mice. The cultured CD34(+) CD133(+) cells had similar engraftment potential to uncultured CD34(+) CD133(+) cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34(+) CD133(+) cells maintain both short-term and long-term HSC activities. To our knowledge, this ~60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.     

Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study, UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin, flt3 ligand, and granulocyte-colony stimulating factor. By week 4-5, we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor, human vascular cell adhesion molecule-1, human intracellular adhesion molecule-1, human CD31, E-selectin, and human macrophage. Furthermore, when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer, better expansion of total nucleated cells, CD34(+) cells, and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells, which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells, we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method, one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors, establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering.     

Extended in vitro expansion of adult, mobilized CD34+ cells without significant cell senescence using a stromal cell coculture system with single cytokine support

The macrophage colony-stimulating factor-deficient bone marrow stromal cell line OP9, derived from osteopetrotic mice, is known to support hematopoietic stem cell (HSC) expansion as well as hematopoietic differentiation of embryonic stem cells. Coculture of HSC in the OP9 system requires cytokine support to achieve significant cell expansion. Recently, we reported extensive expansion without cell senescence of cord blood (CB)-derived HSC cocultured with OP9 stromal cells for more than 18 weeks with a single cytokine support using human thrombopoietin (TPO). In this study, we evaluated the efficiency of the OP9/TPO coculture system to sustain long-term hematopoiesis of adult, granulocyte colony-stimulating factor mobilized human peripheral blood (PB) CD34(+) cells. Maximum cell expansion was attained during the first 4 weeks of coculture. At the same time, the maximum progenitor cell expansion was demonstrated by the production of colony-forming cells and cobblestone area-forming cells. In contrast to the expansion of CB CD34(+) cells, PB CD34(+) cells showed termination of cultures after 8 weeks, independent of the cell expansion rates attained. The evaluation of cell senescence by assessing the telomere length in most cultures showed no relevant telomere shortening, despite rapid decrease in telomerase activity. Interestingly, increases in telomere length were demonstrated. In conclusion, OP9/TPO system provides extensive stem cell expansion without concomitant telomere erosion for both CB and adult CD34(+) cells. Termination of adult CD34(+) cell cocultures seems to be independent of telomere length.     

The impact of progenitor enrichment,serum, and cytokines on the ex vivo expansion of mobilized peripheral blood stem cells: a controlled trial

The aim of this study was to verify, and possibly improve, culture conditions to expand human mobilized peripheral blood stem cells (PBSCs). We investigated the role of three parameters: A) the culture medium (serum-free versus serum-dependent); B) the initial cell population (Ficoll-separated mononucleated cells versus CD34(+)-selected cells), and C) the low concentration of recombinant cytokines, flt3 ligand, and thrombopoietin in association with a basic cocktail of stem cell factor, interleukin (IL)-6, IL-3, GM-CSF, and erythropoietin. Eighteen leukapheresis samples were monitored in static culture for 15 days. The expansion potential was assessed at day 10 and 15 by total nuclear cells, colony-forming-units (CFUs) (burst-forming units-erythroid [BFU-E], colony-forming units-granulocyte-macrophage [CFU-GM], and colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte [CFU-GEMM]), and flow cytometry immunophenotyping (CD34(+)/CD38(-), CD38(+), CD33(+), CD41(+), GlyA(+) progenitor cells). The results, evaluated by multivariate analysis of variance, emphasize that some variables affected the outcome of stem and progenitor cell expansion. CD34(+) enrichment increased expansion of total nuclear cells, number of CD38(+) and CD33(+) late precursors, and number of the CFU-GM compartment. Interestingly, however, quantitative expansion of GlyA(+) and the early progenitor cells (CD34(+)/CD38(-), CFU-GEMM, BFU-E) are favored by the use of unselected mononucleated cells. Regarding the role of serum, no significant difference was observed except for expansion of total nuclear cells, CFU-GM, and BFU-E. Cytokine combinations, in particular the use of flt3 ligand, stimulated expansion of almost all the cellular subsets, reaching a statistical significance for total nuclear cells and CFU-GM. Our study indicates that progenitor and late precursor multilineage cell compartments of mobilized PBSCs may be significantly expanded in short-term cultures by well-defined experimental conditions. Furthermore, these data might be useful when evaluating ex vivo expansion of hematopoietic cells for clinical purposes.