Hematopoietic Support Function Research Articles

Ruxolitinib improves hematopoietic regeneration by restoring mesenchymal stromal cell function in acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a severe complication of allogeneic hematopoietic stem cell transplantation. Hematopoietic dysfunction accompanied by severe aGVHD, which may be caused by niche impairment, is a long-standing clinical problem. However, how the bone marrow (BM) niche is damaged in aGVHD hosts is poorly defined. To comprehensively address this question, we used a haplo-MHC-matched transplantation aGVHD murine model and performed single-cell RNA-Seq of nonhematopoietic BM cells. Transcriptional analysis showed that BM mesenchymal stromal cells (BMSCs) were severely affected, with a reduction in cell ratio, abnormal metabolism, compromised differentiation potential, and defective hematopoiesis-supportive function, all of which were validated by functional assays. We found that ruxolitinib, a selective JAK1/2 inhibitor, ameliorated aGVHD-related hematopoietic dysfunction through a direct effect on recipient BMSCs, resulting in improved proliferation ability, adipogenesis/osteogenesis potential, mitochondria metabolism capacity, and crosstalk with donor-derived hematopoietic stem/progenitor cells. By inhibiting the JAK2/STAT1 pathway, ruxolitinib maintained long-term improvement of aGVHD BMSC function. Additionally, ruxolitinib pretreatment in vitro primed BMSCs to better support donor-derived hematopoiesis in vivo. These observations in the murine model were validated in patient samples. Overall, our findings suggest that ruxolitinib can directly restore BMSC function via the JAK2/STAT1 pathway and, in turn, improve the hematopoietic dysfunction caused by aGVHD.

HIV-1 Tat Protein-Mediated Inflammatory Response Inhibits the Erythroid Hematopoietic Support Function of Bone Marrow Mesenchymal Stem Cells.

Although combination antiretroviral therapy is widely used to treat HIV-1 infection, anemia affects the health and quality of life in a large number of these patients. The proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs), as important support cells in the hematopoietic microenvironment, can be affected by HIV-1 Tat protein. In this study, we explored the mechanism underlying the effect of Tat protein on the hematopoietic support function of BMSCs in erythroid commitment. BMSCs were treated with Tat protein or transfected with Tat mRNA and cocultured with hematopoietic stem cells (HSCs) to detect the number of erythroid colony-forming units (CFUs) and the proportion of mature red blood cells from HSCs. Subsequently, the expression level of a series of erythroid hematopoietic support factors and inflammatory factors in BMSCs after Tat treatment were analyzed. Then, the activation effect of Tat on the mitogen-activated protein kinase/nuclear factor kappa-B (MAPK/NF-κB) pathway, which is an important inflammatory response signaling pathway, was evaluated. The results showed that the number of erythroid CFUs and the production of mature red blood cells supported by BMSCs treated with Tat protein were significantly reduced and the expression of a series of erythroid supporting factors of BMSCs were significantly decreased by Tat protein. Tat-treated BMSCs highly express a variety of inflammatory mediators. Moreover, the expression of P38, p-p38, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, NF-κB, and p-NF-κB was significantly upregulated by Tat protein. In conclusion, Tat protein induces the inflammatory response of BMSCs by activating the MAPK/NF-κB pathway to inhibit the erythroid hematopoietic support function of BMSCs.

Hyperleukocytic Acute Leukemia Circulating Exosomes Regulate HSCs and BM-MSCs.

Hyperleukocytic acute leukemia (HLAL) circulating exosomes are delivered to hematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (BM-MSCs), thereby inhibiting the normal hematopoietic process. In this paper, we have evaluated and explored the effects of miR-125b, which is carried by HLAL-derived exosomes, on the hematopoietic function of HSCs and BM-MSCs. For this purpose, we have isolated exosomes from the peripheral blood of HLAL patients and healthy volunteers. Then, we measured the level of miR-125b in exosomes cocultured exosomes with HSCs and BM-MSCs. Moreover, we have used miR-125b inhibitors/mimic for intervention and then measured miR-125b expression and colony forming unit (CFU). Apart from it, HSC and BM-MSC hematopoietic-related factors α-globulin, γ-globulin, CSF2, CRTX4 and CXCL12, SCF, IGF1, and DKK1 expression were measured. Evaluation of the miR-125b and BAK1 targeting relationship, level of miR-125b, and expression of hematopoietic-related genes was performed after patients are treated with miR-125b mimic and si-BAK1. We have observed that miR-125b was upregulated in HLAL-derived exosomes. After HLAL-exosome acts on HSCs, the level of miR-125b is upregulated, reducing CFU and affecting the expression of α-globulin, γ-globulin, CSF2, and CRCX4. For BM-MSCs, after the action of HLAL-exo, the level of miR-125b is upregulated and affected the expression of CXCL12, SCF, IGF1, and DKK1. Exosomes derived from HLAL carry miR-125b to target and regulate BAK1. Further study confirmed that miR-125b and BAK1mimic reduced the expression of miR-125b and reversed the effect of miR-125b mimic on hematopoietic-related genes. These results demonstrated that HLAL-derived exosomes carrying miR-125b inhibit the hematopoietic differentiation of HSC and hematopoietic support function of BM-MSC through BAK1.

Early Growth Response (EGR)-1 Is a Key Regulator of Human Primary Bone Marrow Stroma Cells with a Dual Role in Proliferation and Hematopoietic Stroma Support Function

Hematopoietic stem cell (HSC) niches are specialized local tissue microenvironments that maintain HSCs and regulate HSC functions. Bone marrow mesenchymal stroma cells (BMSCs) are essential constituents of the HSC niche and they give rise to skeletal lineage cells as well as the hematopoietic stroma. Despite recent progress in the identification of the primary bone fide BMSC in human bone marrow, knowledge regarding the regulation of BMSC proliferation, differentiation and hematopoietic support function is incomplete.We have previously shown that the expression of Early Growth Response 1 (EGR1) in primary BMSCs is substantially higher compared to the non-colony-forming cells and cultured stromal cells, respectively, indicating a possible role of EGR1 in BMSC regulation (Li et al., Stem Cell Reports, 3(6):965-74, 2014). EGR1 encodes a zinc finger transcription factor that exemplifies a group of immediate early response genes, which have diverse functions in cell growth, development and stress response. EGR1 regulates murine HSC proliferation and localization (Min et al., Cell Stem Cell, 2(4):380-91, 2008), but its role in stroma cell regulation has thus far not been investigated. This study therefore aimed to investigate the functional role of EGR1 in human primary BMSCs.Quantitative gene expression analysis demonstrated that EGR1 expression was 126.9 ± 9.1-fold and 2.8 ± 0.2-fold higher in highly CFU-F (colony-forming unit, fibroblast) enriched human primary linneg/CD45neg/CD271pos/CD140aneg bone marrow cells compared to the non-colony forming CD45neg/CD271neg cell population and CD45neg/CD271pos/CD140apos cells with minimum CFU-F activity. Furthermore, we confirmed that EGR1 expression in cultured cells was down-regulated in comparison with primary BMSCs.Multi-lineage differentiation capacity and surface marker expression profiles of BMSCs were not affected by shRNA-induced EGR1 down-regulation. However, colony-forming capacity was enhanced (1.9 ± 0.1-fold) and population doubling times were reduced by EGR1 knockdown compared to scramble controls. Furthermore, the HSC stroma supporting function as measured by production of transplantable HSC in co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×104 CD34pos seeded cord blood cells) was reduced when EGR1 knockdown cells were used as stroma layer.On the other hand, whereas CFU-F formation was virtually absent in EGR1 overexpressing cells and population doubling times increased by 2.4 ± 0.3-fold, the hematopoietic stroma support function was considerably improved by EGR1 overexpression as indicated by increased percentages and absolute numbers of transplantable CD34posCD90pos cells produced in co-cultures with EGR1 overexpressing BMSCs as stromal cells (12,496 ± 887 for EGR1 overexpression vs 6,867 ± 1242 for GFP control). Accordingly, CD34pos cells expanded on EGR1 overexpressing stroma exhibited improved in-vivo long-term reconstitution levels in NSG mice compared with cells grown on control stroma.To investigate how EGR1 regulates proliferation and hematopoiesis support in BMSCs, transwell experiments were performed showing that both soluble factors and cell-to-cell contact were important for EGR1-mediated hematopoiesis support. Furthermore, proteomic analysis revealed that a group of proteins related to oxidative-reduction processes was downregulated whereas reactive oxygen species (ROS) levels were elevated in EGR1 knockdown cells. In addition, gene expression profiling analysis identified a number of hematopoietic support genes that were upregulated in EGR1 overexpressing cells compared with control cells.In summary, our data indicate that EGR1 is an important BMSC regulator with a dual role on proliferation and hematopoietic stroma support function. EGR1 expression negatively regulated BMSC proliferation while positively regulating hematopoietic support function, which points to its possible role in coordinating the specific functions of BMSC in their different biological contexts. DisclosuresNo relevant conflicts of interest to declare.

Long-Term Clearance of Senescent Cells Prevents the Hematopoietic Stem Cell Aging in Naturally Aged Mice

Senescent cells (SnCs) accumulate with age. Genetic or pharmacological clearance of SnCs delays several age-associated disorders and extends the healthspan of both progeroid and wild-type (WT) mice (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). In addition, our previous study showed that transient depletion of SnCs could rejuvenated aged hematopoietic stem cells (HSCs) and muscle stem cells in normally aged mice (Chang J et al. Nat Med, 22:78, 2016). These findings suggest that SnCs play a causative role in aging and certain aging-related pathologies, and that selective clearance of SnCs with a senolytic agent that can selectively kill SnCs can be beneficial in part by rejuvenating aged tissue stem cells. Thus, senolytic drugs are potential novel anti-aging agents. However, SnCs can re-accumulate within a few weeks after depletion in mice, suggesting that long-term and intermittent clearance of SnCs is required to extend healthspan, as shown in previous studies (Baker DJ et al. Nature, 479: 232, 2011; Baker DJ et al. Nature, 530: 184, 2016). It is not known how long-term and intermittent clearance of SnCs affects tissue stem cells such as HSCs because HSCs are mostly quiescent in order to self-renew and prevent exhaustion. We used p16-3MR transgenic mice to evaluate the effect on HSCs of long-term and intermittent clearance of SnCs by ganciclovir (GCV), which selectively kills SnCs in these mice. We treated WT and p16-3MR mice with GCV (25 mg/kg/day x 5 days/per cycle with a 2-week interval between the cycles by ip injection) or saline (vehicle control) from 12 to 24 months of age. GCV effectively prevented SnC accumulation in p16-3MR mice but had no effect in WT mice. In general, aged p16-3MR mice treated with GCV looked healthier than old vehicle-treated p16-3MR mice and old WT mice treated with vehicle or GCV as expected (Baker DJ et al. Nature, 530: 184, 2016). Importantly, GCV abrogated aging-induced increases in p16 expression, p38MAPK activation, and accumulation of DNA double-strand breaks in HSCs in p16-3MR mice. This abrogation was associated with a significant improvement in HSC clonogenicity, suggesting that long-term and intermittent clearance of SnCs prevents HSC aging in naturally aged mice. This suggestion is confirmed by competitive serial and limited dilution HSC transplantation assays, which revealed that HSCs from GCV-treated old p16-3MR mice were functionally indistinguishable from those of young mice. To elucidate the mechanism by which SnC clearance prevents HSC aging, we evaluated the clonogenicity of Lin−Sca1+c-Kit+ (LSK) cells from young or vehicle- or GCV-treated old p16-3MR mice in co-cultures with bone marrow stromal cells (BMSCs) from these mice. We found that clearance of SnCs not only prevented the decline in HSC clonogenic function with aging but also preserved the hematopoietic supporting function of BMSCs. This preservation was associated with reduced levels of Cdkn2a and Cdkn1a mRNAs and senescence-associated secretory phenotype (SASP) factors (such as Il1β, Tnfα and Ccl5), which can adversely affect HSC self-renewal, and decrease of adipogenic and osteoclastic differentiation. In addition, the levels of the HSC self-renewal promoting factor Spp1 mRNA was higher in BMSCs from GCV-treated old p16-3MR mice than in the cells from vehicle-treated old p16-3MR mice. These findings suggest that long-term and intermittent clearance of SnCs can also inhibit BMSC senescence, which contributes to the prevention of HSC aging. Disclosures He: University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Chang:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Kim:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Almeida:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Campisi:Unity Biotechnology: Equity Ownership, Other: co-founder of Unity Biotechnology which develops small-molecule senolytic drugs. Zhou:University of Arkansas for Medical Sciences: Patents & Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents.

Transplanted spleen stromal cells with osteogenic potential support ectopic myelopoiesis.

Spleen stromal lines which support in vitro hematopoiesis are investigated for their lineage origin and hematopoietic support function in vivo. Marker expression and gene profiling identify a lineage relationship with mesenchymal stem cells and perivascular reticular cells described recently in bone marrow. Stromal lines commonly express Cxcl12, Pdgfra and Pdgfr typical of bone marrow derived perivascular reticular cells but reflect a unique cell type in terms of other gene and marker expression. Their classification as osteoprogenitors is confirmed through ability to undergo osteogenic, but not adipogenic or chondrogenic differentiation. Some stromal lines were shown to form ectopic niches for HSCs following engraftment under the kidney capsule of NOD/SCID mice. The presence of myeloid cells and a higher representation of a specific dendritic-like cell type over other myeloid cells within grafts was consistent with previous in vitro evidence of hematopoietic support capacity. These studies reinforce the role of perivascular/perisinusoidal reticular cells in hematopoiesis and implicate such cells as niches for hematopoiesis in spleen.

HIV-1 Tat protein inhibits the hematopoietic support function of human bone marrow mesenchymal stem cells

Most HIV-1-infected patients experience hematopoiesis suppression complications. Bone marrow mesenchymal stem cells (BMSCs) are involved in regulation of hematopoietic homeostasis, so we investigated the role of Tat, a protein released by infected cells in bone marrow and impacted differentiation potential of mesenchymal stem cells, in the BMSC hematopoietic support function. BMSCs were treated with HIV-1 Tat protein (BMSCTat-p), transfected with HIV-1 Tat mRNA (BMSCTat-m) or treated with solvent (PBS) (BMSCcon) for 20 days. Then, the hematopoietic support function of BMSCTat-p, BMSCTat-m and BMSCcon was analyzed via ex vivo expansion of hematopoietic stem cells (HSCs) grown on the BMSCs and via in vivo cotransplantation of HSCs and BMSCs. In addition, the hematopoiesis-supporting gene expression patterns of BMSCTat-p, BMSCTat-m and BMSCcon were compared. The results showed that BMSCTat-p and BMSCTat-m displayed reduced expansion, a decline in the number of colony forming units (CFUs) and a decreased proportion of the primitive subpopulation of hematopoietic stem cells under coculture conditions compared with BMSCcon. The ability of BMSCTat-p to support hematopoietic recovery was also impaired, which was further confirmed by the patterns in gene expression analysis. In conclusion, Tat treatment reduced the function of BMSCs in hematopoietic support, likely by downregulating the expression of a series of hematopoietic cytokines.

Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells.

Human bone marrow stromal cells (BMSC) are key elements of the hematopoietic environment and they play a central role in bone and bone marrow physiology. However, how key stromal cell functions are regulated is largely unknown. We analyzed the role of the immediate early response transcription factor EGR1 as key stromal cell regulator and found that EGR1 was highly expressed in prospectively-isolated primary BMSC, down-regulated upon culture, and low in non-colony-forming CD45neg stromal cells. Furthermore, EGR1 expression was lower in proliferative regenerating adult and fetal primary cells compared to adult steady-state BMSC. Overexpression of EGR1 in stromal cells induced potent hematopoietic stroma support as indicated by an increased production of transplantable CD34+CD90+ hematopoietic stem cells in expansion co-cultures. The improvement in bone marrow stroma support function was mediated by increased expression of hematopoietic supporting genes, such as VCAM1 and CCL28. Furthermore, EGR1 overexpression markedly decreased stromal cell proliferation whereas EGR1 knockdown caused the opposite effects. These findings thus show that EGR1 is a key stromal transcription factor with a dual role in regulating proliferation and hematopoietic stroma support function that is controlling a genetic program to co-ordinate the specific functions of BMSC in their different biological contexts.

Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review.

SummaryIdentified 50 years ago, mesenchymal stromal/stem cells (MSCs) immediately generated a substantial interest among the scientific community because of their differentiation plasticity and hematopoietic supportive function. Early investigations provided evidence of a relatively low engraftment rate and a transient benefit for challenging congenital and acquired diseases. The reasons for these poor therapeutic benefits forced the entire field to reconsider MSC mechanisms of action together with their ex vivo manipulation procedures. This phase resulted in advances in MSCs processing and the hypothesis that MSC‐tissue supportive functions may be prevailing their differentiation plasticity, broadening the spectrum of MSCs therapeutic potential far beyond their lineage‐restricted commitments. Consequently, an increasing number of studies have been conducted for a variety of clinical indications, revealing additional challenges and suggesting that MSCs are still lagging behind for a solid clinical translation. For this reason, our aim was to dissect the current challenges in the development of still promising cell types that, after more than half a century, still need to reach their maturity. stem cells translational medicine 2019;8:1135–1148

In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line.

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.

HIV-1 p55-gag protein induces senescence of human bone marrow mesenchymal stem cells and reduces their capacity to support expansion of hematopoietic stem cells in vitro.

Patients with human immunodeficiency virus-1 (HIV-1) infection often present with hematopoietic failure. As the important hematopoietic support cells in the bone marrow (BM), the BM mesenchymal stem cells (BMSCs) can be impacted by HIV proteins that are released by infected cells within BM. In this study, we tested whether HIV protein p55-gag could induce senescence of BMSCs and reduce their capacity to support expansion of hematopoietic stem cells in vitro. BMSCs were chronically treated with p55-gag (BMSCgag ) for up to 20 days, and their proliferative activity and senescence makers were compared to nontreated cells (BMSCcon ). Then, we analyzed the hematopoietic support function of BMSCcon and BMSCgag by determining cellular proliferation, colony-forming ability, and primitive hematopoietic populations of hematopoietic progenitors grown on the BMSCs. In addition, we compared the gene expression patterns for supporting hematopoiesis of BMSCcon and BMSCgag. The results show that when compared to BMSCcon , BMSCgag reduced their proliferative activity and underwent senescence. The ability of BMSCgag to support the expansion of committed hematopoietic progenitors from umbilical cord blood-derived CD34+ cells may be impaired, while the expression of genes associated with maintaining and enhancing hematopoiesis appeared to be decreased in treated BMSCs compared to control BMSCs. In conclusion, senescence induced by p55-gag resulted in decreased hematopoietic support function of BMSCs through reducing a series of hematopoietic cytokine expression.

Early Growth Response (EGR)-1 Expression Regulates Colony Forming Capacity and Hematopoietic Support Function in Human Primary Bone Marrow Stromal Stem Cells

Bone marrow stromal stem cells (BMSCs) are essential components of the hematopoietic environment. BMSCs play a key role in regulating hematopoiesis and, furthermore, as skeletal stem cells they give rise to osteoblasts, adipocytes, and chondrocytes. However, despite this important role of BMSCs in bone and bone marrow physiology, little is known about how proliferation, differentiation and hematopoietic support functions of BMSCs are regulated. We hypothesized that primary human BMSCs have a distinct transcriptional regulatory system, which control BMSC stem cell properties and biological functions. We have previously reported gene expression profiling of highly-enriched human primary BMSCs (Li et al, Stem Cell Reports, 3(6):965-74, 2014), which demonstrated a substantially higher expression of early growth response 1 (EGR1) in primary cells compared to the non-colony-forming cells and cultured stromal cells, respectively. EGR1 is a member of the immediate early response transcription factor family, which has a function in cell growth, development, and stress responses in many tissues. EGR1 has been previously reported to be important for hematopoietic stem cell (HSC) proliferation and localization (Min et al. Cell Stem Cell, 2(4):380-91, 2008), but its role in non-hematopoietic bone marrow cells has thus far not been investigated. Therefore, we aimed to study the possible role of EGR1 in stroma stem cell proliferation and hematopoietic supporting function.Our data demonstrate that the expression of EGR1 as measured by qPCR was 126 ± 9-fold higher in highly fibroblast colony-forming cells (CFU-F) enriched human primary linneg/CD45neg/CD271pos/CD140aneg bone marrow cells compared to the non-colony forming CD45neg/CD271neg cell population. Furthermore, EGR1 expression in CD271posCD140aneg cells was 3 ± 0.2 -fold higher than in the CD271posCD140apos cell population, which has only minimal CFU-F activity. EGR1 expression decreased dramatically during culture with a more than 30-fold difference between primary and passage one and six cells. Down-regulation of EGR1 expression by shRNA did not affect the multi-differentiation capacity (adipogenic, osteogenic) and surface marker expression profile of BMSCs in vitro compared to controls. However, colony-forming capacity and proliferation was considerably increased in EGR1 knockdown cells, i.e. shRNA- transduced stromal cells produced up to 1.8 ± 0.1-fold more CFU-F compared to controls, whereas CFU-F were virtually absent when assaying EGR1 overexpressing cells. Furthermore, population doubling times were decreased in EGR1 knockdown cells but significantly increased (2.4 ± 0.3-fold) in EGR1 overexpressing cells. These data indicate that EGR1 expression negatively regulates BMSC proliferation and colony-forming capacity. On the other hand, hematopoietic support function was decreased in EGR1 knockdown cells as measured by the production of transplantable CD34posCD90pos HSC in stroma co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×104 CD34pos seeded cord blood cells). Here, the positive effect of the supporting stroma was neutralized by knockdown of EGR1. Numbers of CD34posCD90pos HSC produced in co-cultures with EGR1 knockdown stroma cells were as low as 1,053 ± 316 compared to 6,100 ± 840 in control co-cultures (scamble control). Without stroma, 840 ± 210 CD34posCD90pos cells were generated from 1×104 seeded CD34pos cells. Furthermore, expansion of CD34posCD90posCB cells was increased in co-cultures with EGR1 overexpressing cells, indicating that EGR1is a positive regulator of hematopoietic stroma support in human BMSC, and confirmatory in-vivo xenotransplantation studies are ongoing.In summary, EGR1 is highly expressed by primary BM-MSC compared with non-colony forming cells and is downregulated during culture. EGR1 expression negatively regulates BMSC proliferation and colony formation while positively regulating hematopoietic stroma support function. Our data thus indicate that EGR1 may act as an important BMSC regulator coordinating the specific functions of BMSC in their different biological contexts. DisclosuresNo relevant conflicts of interest to declare.

Leukemia-Induced Dysregulation of Bone Marrow Skeletal Stem Cells (SSC) Subpopulations and Their Hematopoietic Supportive Function

Specialized bone marrow microenvironment (BMM) niches are essential for hematopoietic stem cell (HSC) maintenance and differentiation, and include perivascular cells with mesenchymal stem cells (MSC) capacity. The role of BMM niches in regulating LSC growth remains unclear. We have shown that cytokines altered in CML BMM contribute a competitive growth advantage to CML LSC over normal HSC (Zhang et al. Cancer Cell. 2012; 21(4):577). However, the effect of leukemic hematopoiesis on BM MSC and their progeny, and the effect of such alterations on the regulation of LSC versus HSC growth, is not well studied. Recently rigorous single-cell analyses and lineage tracing has been used to delineate a BM skeletal stem cell (SSC) hierarchy (Chan et al. Cell. 2015, 160(1-2):285). Eight distinct subpopulations were identified within the CD45-Ter119-Tie2-AlphaV+ BM mesenchymal cells, of which the mSSC (Thy-6C3-CD105-CD200+) subpopulation generated all of the other subpopulations. Two multipotent progenitor populations, pre-BCSP (Thy-6C3-CD105-CD200-) and BCSP (Thy-6C3-CD105+) gave rise to bone, cartilage and hematopoietic supportive stroma. The 6C3 (Thy-6C3+CD105+) and HEC (Thy-6C3+CD105-) populations gave rise to stroma, the BLSP (Thy+6C3-CD105-) and Thy (Thy+6C3-CD105+CD200-); populations formed bone, and the PCP (Thy+6C3-CD105+CD200+) population formed cartilage. To examine how these mesenchymal populations related to those targeted in the Cre-transgenic mouse lines commonly used to study BMM, we performed lineage tracing by crossing Prx-1-Cre, Sp7-Cre, BGLAP-Cre, and UBC-Cre mice with Rosa26CAG-tdTomato mice, in which tdTomato is expressed by cells targeted for Cre-mediated recombination. Interestingly, mSSC and 6C3 cells were targeted in all Cre lines, while the Thy population was only targeted in Prx1-Cre mice. We examined SSC population distribution and function following CML development using the SCL-tTA-BCR-ABL transgenic mouse model. The frequency and absolute number of bone-forming Thy (WT: 442.5±65.66; CML: 2501±705.5; p=0.012, n=14) and stroma-forming 6C3 (WT:1061±193.7; CML: 2271±358.5; p=0.009, n=14) cells were significantly increased in BM of CML mice compared to wild-type (WT) mice, while primitive mSSC, pre-BCSP and BCSP populations remained unchanged. Expression of major hematopoietic regulatory molecules, transcription factors and cell cycle regulatory genes in CML and WT BMM subsets was measured by qPCR analysis using the Fluidigm system. Integrin α4 and Tenascin levels were reduced, whereas PDGFRβ, BMP2 and Hif-1α levels were increased in CML compared to normal mSSC. Several WNT pathway members were significantly upregulated in CML compared to WT 6C3 stromal cells, whereas CXCL12, KitL, IL-3, and IGF-1 were reduced. Expression of cell cycle regulators, Hedgehog and Notch pathway members remained unchanged. In ongoing experiments to investigate whether these subsets have altered HSC supportive capacity towards normal and CML stem cells, we co-cultured FACS sorted normal or CML LTHSC with FACS sorted normal or CML mSSC, BCSP, 6C3, and Thy cells for 3 days followed by transplantation into lethally irradiated WT mice. Normal LTHSC engraftment at 4 weeks post-transplant was enhanced by coculture with normal mSSC but not with CML mSSC. In contrast, there was increased engraftment of normal LTHSC co-cultured with CML compared to normal BCSP, Thy or 6C3 cells. CML LTHSC engraftment at 4 weeks was not enhanced by either normal or CML mSSC, but was enhanced following co-culture with normal as well as CML Thy and 6C3 cells. These results indicate that SSC subpopulations from CML BM demonstrate altered capacity to support normal LTHSC compared to their normal counterparts, but that CML and WT SSC populations may have similar effects on CML LTHSC. This experiment is ongoing with results of longer-term engraftment pending. In conclusion, we have further characterized a phenotypically-defined skeletal stem cell hierarchy and identified significant alterations in distribution of SSC subpopulations within CML compared to WT BM, with significantly altered expression of important hematopoietic regulatory molecules and HSC supportive function. We hypothesize that leukemia-induced SSC alterations may confer a growth advantage to CML LSC over normal HSC, and that characterization of key regulatory mechanisms could facilitate therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Effects of mesenchymal stem cells on hematopoietic support and secretory function of T lymphocytes in patients with aplastic anemia

Effects of mesenchymal stem cells on hematopoietic support and secretory function of T lymphocytes in patients with aplastic anemia

Contribution of Leukemia-Induced Alterations in Mesenchymal Cell Subpopulations to Altered Regulation of Leukemic and Normal Stem Cells in the CML BM Microenvironment

Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem cell (HSC) maintenance. The function of microenvironmental niches in regulating leukemia stem cell (LSC) growth is not well understood. We have reported that alterations in chemokine and cytokine expression in the CML BM microenvironment, including reduction in expression of the critical hematopoietic chemokine CXCL12, may differentially alter CML and normal stem cell localization and growth, and provide a competitive growth advantage to CML LSC. Here we evaluated the contribution of leukemia-induced alterations in BM mesenchymal cell subpopulations to altered BM microenvironmental function in CML. Our group has used cell surface markers to characterize mesenchymal cells in the BM of adult mice. These studies have identified three distinct osteoprogenitor subpopulations [CD166+CD146- (CD166+);CD166-CD146+ (CD146+); and CD166-CD146-Sca1+ (Sca1+) cells] as well as a phenotypically defined population of CXCL12-adundant reticular (CAR) cells (Sca1-CD44+CD51+CD106+CD140a+) within CD45-Ter119-CD31- BM mesenchymal cells. We have shown that Sca1+ cells represent primitive mesenchymal progenitors that can generate the other mesenchymal populations, whereas CD146+ cells may represent an intermediate population between Sca1+ cells and CD166+ osteoblasts. Using the SCL-tTA-BCR/ABL mouse model of CML, we examined leukemia-induced alterations in distribution and LTHSC supportive function of mesenchymal cells in CML BM. We observed that the frequency of Sca1+ (p=0.001) and CD146+ (p=0.02) progenitors were significantly increased, and of CD166+ osteoblastic cells (p<0.05) and CAR (p<0.02) cells were significantly reduced, in CML compared to normal BM. CD146+ and CD166+ cells, but not Sca1+ cells, from CML BM showed reduced proliferation in vitro compared to their normal counterparts. To evaluate hematopoietic supportive function, selected normal and CML mesenchymal subpopulations were cocultured with CD45.1 normal and CML LTHSC for 3 days at a 1:1 ratio. LTHSC were then transplanted into CD45.2 recipient mice (200 normal LTHSC/mouse; 1000 CML LTHSC/mouse). Normal LTHSC cocultured with normal Sca1+ (p<0.03), CD146+ (p<0.02), CD166+ (p<0.02), and CAR (p<0.03) cells demonstrated significantly increased hematopoietic engraftment in recipient mice at 16 weeks compared with control LTHSC cultured without mesenchymal cells. In contrast engraftment of normal LTHSC co-cultured with CML compared to normal BM mesenchymal subsets was significantly reduced [Sca1+ (p<0.03), CD146+ (p<0.02), CD166+ (p=0.06), and CAR (p<0.02) cells]. CML LTHSC cultured with normal Sca1+ (p<0.002), CD146+ (p<0.001), CD166+ (p<0.001), and CAR (p<0.03) cells showed significantly increased engraftment in recipient mice after transplantation and increased leukemia development compared with CML LTHSC cultured without stromal cells. CML LTHSC showed similar engraftment in recipient mice after coculture with CML and normal Sca1+ and CD146+ cells, but significantly reduced engraftment after coculture with CML compared to normal CD166+ (p<0.01) and CAR (p<0.02) cells. In conclusion, our studies have identified leukemia associated alterations in the distribution of mesenchymal subpopulations in the CML BM microenvironment, with decrease in more differentiated CD166+ osteoblastic cells and CAR cells and increase in primitive Sca-1+ and CD146+ cells. The reduction in CXCL12 producing CAR cells, and the reduced ability of CML Sca1+ and CD146+ cells to support normal compared to CML LTHSC, may provide a competitive growth advantage to the leukemic clone within the CML BM microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Senescence of bone marrow mesenchymal stromal cells is accompanied by activation of p53/p21 pathway in myelodysplastic syndromes.

The contribution of bone marrow mesenchymal stromal cells (BMMSCs) to the pathogenesis of myelodysplastic syndrome (MDS) has created controversies. In this study, we confirmed that BMMSCs from MDS patients showed prominent features of senescence, which were characterized by increased cell size, decreased proliferation and colony-forming potential, alteration of cytoskeleton, and increased senescence-associated β-galactosidase (SA-β-Gal) activity. Interestingly, the apoptosis assay results showed that the percentage of apoptosis cells was very low and the difference was not significant between MDS patients and normal controls. Moreover, the osteogenic differentiation potential of BMMSCs from lower risk but not higher risk MDS was impaired, indicated by cytochemical stainings and reduced expressions of RUNX2. In addition, BMMSCs from MDS patients had impaired hematopoietic supporting function. Furthermore, the expression of p53 and p21 which played an important role in regulating the senescence progress of BMMSCs was significantly increased, whereas levels of p16 and pRb expression were not changed in the BMMSCs from MDS patients. Taken together, our comprehensive analysis shows that BMMSCs from MDS patients exhibited senescent behavior and activation of p53/p21 pathway probably played an important role in the senescence process.

Pluripotent Fibrosis Initiating Mesenchymal Stromal Cell Linked To Cytopenias In LGL Leukemia Through Genetic Reprogramming

BackgroundLarge Granular Lymphocyte (LGL) leukemia is associated with chronic lymphoproliferation in association with unexplained cytopenias. Discovery of somatic mutations in STAT3 provides new insights into the molecular basis of LGL leukemia, but the precise mechanism leading to cytopenias remains unresolved. To gain insight into this mechanism, bone marrow fibrosis (Fig. 1A) was found to occur in 88% of cases and is significantly associated with splenomegaly, the presence of cytopenias, and co- existence of autoimmune diseases. In this study, we are reporting the identification of the fibrosis initiating cell population. [Display omitted] MethodsPrimary mesenchymal stromal cultures (MSCs) from LGL leukemia BM were isolated and grown under reduced oxygen conditions using undifferentiated and differentiation-specific culture conditions (Fig. 1B). These cultured cells were confirmed to deposit greater amounts of fibrillar Type I, III and V collagen matrix consistent with the heavy reticulin and trichrome positive stains observed in bone marrow biopsies (Fig. 1C). This collagen-producing cell population failed to support normal hematopoietic progenitor proliferation and colony formation in co-culture assays. Microarray analysis of the collagen-producing mesenchymal cell population was conducted and pathway analysis was performed using the GeneGo analysis platform from MetaCore™. Each list of genes was individually grouped and evaluated for membership on the set of GeneGo® pathway maps under the functional ontology enrichment tool. Calculation of p-values using hypergeometric distribution was used to determine pathway statistical significance. Flow cytometry was conducted to further evaluate the mesenchymal lineage. ResultsSupervised hierarchical clustering was performed to determine the overall similarity of LGL MSCs to publicly available, platform-compatible, microarrays from cells of mesenchymal lineage. Clustering showed that both patient and control MSCs display expression patterns distinct from committed mesenchymal lineages including osteoblasts, chondrocytes, and adipocytes. Mesenchymal-derived fibroblast differentiation can be distinguished by down-regulated CD29, CD44, CD105, CD106, CD117, bone morphogenetic protein receptor, and Sca-1 expression, and by up-regulated collagen type I, collagen type III, tenacin C, fibronectin, matrix metalloproteinase 1, fibroblast-specific protein 1, and vimentin. By microarray analysis and flow cytometry, we find no evidence that the cells are differentiated cells aside from collagen over expression. In order to define their pluripotent potential, sub-confluent cultures were grown in three types of differentiating media. Primary MSCs from both LGL leukemia patients and healthy controls were similar in their capacity to undergo trilineage differentiation into osteoblasts (stained with Alizarin Red S), adipocytes (Oil red O), and chondrocytes (Alican blue) (Fig. 1D). Next, pathway analyses revealed significant re-programming of the pluripotent MSCs. ECM production pathways were increased while proliferation pathways were down-regulated. Proliferation of MSCs is governed by production of autocrine growth factors. Both leukemia inhibitor factor (LIF) and basic fibroblast growth factor (FGFb) were significantly reduced compared to control MSCs (p<0.001) and the proliferative capacity of the LGL MSCs was severely diminished. Exogenous FGFb, but not LIF, restored LGLL MSC proliferation comparable to normal MSCs (Fig. 1E), normalized their spindle-shaped morphology, and maintained MSC plasticity. Interestingly, treatment with exogenous FGFb normalized collagen deposition (Fig. 1F) and restored hematopoietic supporting function. ConclusionSTAT-activated LGL leukemia cells, like myeloproliferative neoplasms, share a propensity for the development of bone marrow fibrosis. These results indicate that MSCs from LGL leukemia are pluripotent, but are re-programmed to deposit excessive amounts of collagen. Restoration of FGFb maintains the pluripotency of these MSCs while reversing the fibrosis phenotype. Our data suggests that FGFb-cultured MSCs may be used to reverse cytopenias in LGL leukemia. Disclosures:No relevant conflicts of interest to declare.

Understanding the role of the microenvironment during definitive hemopoietic development

Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow have identified osteoblasts and endothelial cells as two important supportive cell types within the niche and demonstrated that interactions between HSCs and cellular and extracellular components within the endosteal and perivascular regions are critical for HSC regulation. However, the understanding of the role of the microenvironment in definitive HSC establishment, expansion, and maintenance during embryonic development is extremely limited. This review focuses on what is known about the components of each HSC microenvironment at various developmental stages and their known functional roles.

Molecular and functional characterization of human bone marrow adipocytes

Adipocytes are a cell population largely located in the human bone marrow cavity. In this specific microenvironment where adipocytes can interact with a variety of different cells, the role of fat is mainly unknown. To our knowledge, this report is the first to characterize mature adipocytes isolated from human bone marrow (BM-A) molecularly and functionally to better understand their roles into the hematopoietic microenvironment. Healthy BM-A were isolated after collagenase digestion and filtration. We studied the morphology of BM-A, their gene expression and immunophenotypic profile and their functional ability in the hematopoietic microenvironment, comparing them with adipocytes derived from adipose tissue (AT-A). BM-A showed a unilocular lipid morphology similar to AT-A and did not lose their morphology in culture; they showed a comparable pattern of stem cell-surface antigens to AT-A. In line with these observations, molecular data showed that BM-A expressed some embryonic stem cells genes, such as Oct4, KLf4, c-myc, Gata4, Tbx1, and Sox17, whereas they did not express the stem cell markers Sox2 and Nanog. Moreover, BM-A had long telomeres that were similar to bone marrow mesenchymal stem cells. Notably, BM-A supported the survival and differentiation of hematopoietic stem cells in long-term cultures. These results showed that BM-A are stromal cells with a gene expression pattern that distinguished them from AT-A. BM-A showed stem cell properties through their hematopoietic supporting function, which was certainly linked to their role in the maintenance of the bone marrow microenvironment. Depending on specific demands, BM-A may acquire different functions based on their local environment.

Human Umbilical Cord Blood-Derived Stromal Cells Are Superior to Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Inducing Myeloid Lineage Differentiation In Vitro

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.