Mesenchymal Stromal Cells Support Research Articles

Mitochondrial transfer mediates endothelial cell engraftment through mitophagy.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.

Targeting S100A9 in the Inflammatory Myelodysplastic Hematopoietic Niche Reprograms the Functional Properties of CD271+ Mesenchymal Stromal Cells

Myelodysplastic syndromes (MDS) belong to the most common hematological malignancies in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. An involvement of the bone marrow microenvironment (BMME) in disease initiation and progression is indisputable. The profound immune dysregulation and sustained inflammation, essentially mediated by mesenchymal stromal cells (MSCs), are further factors contributing to ineffective hematopoiesis and driving disease progression. The alarmin S100A9 is a TLR4 and CD33 ligand that acts as key player for the inflammasome activation in the MDS BM. The novel small molecular drug Tasquinimod (TASQ, Active Biotech) is a S100A9 inhibitor and has demonstrated antitumor and immunomodulatory properties in a broad range of solid tumors; however, little is known about its effects in myeloid malignancies. Here, we aim to decipher the effects of TASQ on MDS MSC subpopulations in more detail. Therefore, MSCs were isolated from BM aspirates ('asp-MSCs', n=12) or biopsies ('bp-MSCs', n=5) of patients with either low-risk MDS, CMML or age-adjusted healthy donors and were exposed to TASQ (10 µM) in vitro. Flow cytometry analysis of the MSC markers CD44, CD73, CD90, CD105, and CD146 did not show significant differences in neither asp- nor bp-MSCs after treatment. Interestingly, CD271, which is hard to detect in asp-MSCs, was decreased between 40% and 70% in bp-MSCs after TASQ treatment. This was accompanied by an inhibited expression of the checkpoint inhibitor PD-L1. The MDS BM has a higher potency for adipogenic differentiation which further supports the inflammatory conditions and the suppression of normal hematopoiesis. Treatment with TASQ resulted in about 50% inhibition of adipogenesis as demonstrated by significantly lower expression of adipocyte protein 2 in differentiated asp- and bp-MSCs. To investigate possible metabolic modulations of MSCs by TASQ, the oxygen consumption rate was measured as a readout of mitochondrial function by using the Cell Mito Stress Test (Agilent Technologies) in a XF96 analyzer. The basal and the maximal respiratory rate as well as ATP production were increased in TASQ-treated bp-MSC cultures suggesting a positive effect for their bioenergetic capacity. This was supported by detection of higher levels of glutamate dehydrogenase 1, a key enzyme in glutaminolysis converting glutamate to α-ketoglutarate for entry into the TCA cycle, and of the glutamine transporter Slc1a3 in TASQ-treated cells. NMR analyses revealed increased levels of glutamate and citrate as well as of several lipids, such as free cholesterol LDL, phospholipids LDL-6 and Apo-B LDL6 in supernatants of bp-MSCs after TASQ treatment. To investigate the impact of TASQ on CD271 expression in more detail, we sorted CD45-/CD73+/CD90+/CD271+/- bp-MSCs. Gene expression analysis by RNA sequencing of sorted MSCs revealed differential expression of several pathways, e.g. an enrichment of inflammation related molecules in the CD271+ samples, whereby TASQ could abrogate this effect. Moreover, a cytokine array analysis of culture supernatants of sorted cells showed enhanced IFNg, IL1b and IL-6 secretion of CD271+ MSCs compared to CD271- and TASQ-treated cells. To evaluate the impact on the hematopoietic support of MSCs, we analyzed CD271-sorted bp-MSC co-cultures with CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed a significantly decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay for CD271+ MSCs, nominating this MSC subpopulation as responsible for the diminished hematopoietic support. Interestingly, the number of CAF-C and CFU-E/CFU-GM could be increased by TASQ pre-treatment of the stromal layer. In conclusion, CD271+ MSCs play a crucial role in the inflammatory MDS BMME. The inhibition by TASQ efficiently blocks adipogenic differentiation and secretion of pro-inflammatory cytokines, thereby modulating the MSC energetic profile and enhancing their potential to support hematopoiesis in vitro.

Targeting the Inflammatory Niche in MDS By Tasquinimod Restores Hematopoietic Support and Suppresses Immune-Checkpoint Expression in Vitro

IntroductionMyelodysplastic syndromes (MDS) belong to the most common hematological neoplasms in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. There is increasing evidence that an aberrant innate immune response and a proinflammatory bone marrow (BM) microenvironment play a critical role in the pathogenesis of MDS. The alarmin S100A9, a key player for regulation of inflammatory responses, has been shown to be elevated in MDS patients. It directs an inflammatory cell death (pyroptosis) by increased NF-kB mediated transcription and secretion of proinflammatory, hematopoiesis-inhibitory cytokines and production of reactive oxygen species.Tasquinimod (TASQ, Active Biotech) is a novel, oral small molecular drug with S100A9 inhibitory activity and it is currently investigated in a phase Ib/IIa trial in relapsed/refractory multiple myeloma (NCT04405167). TASQ has demonstrated anti-angiogenic, antitumor and immunomodulatory properties in a broad range of preclinical solid tumor models; however, little is known about its effects in myeloid malignancies.AimWe investigated the role of S100A9 in cellular models of MDS and the potential of TASQ to target S100A9 within the MDS stroma in vitro.MethodsImmunohistochemical staining of S100A9, CD271+ mesenchymal stromal cells (MSCs), CD68+ macrophages and CD66b+ neutrophils in BM tissues from MDS patients and healthy donors was performed with multiplex immunohistochemistry and analyzed with the VECTRA imaging system.MSCs from patients with either low-risk MDS, CMML or age-adjusted healthy donors were exposed to S100A9 (1.5µg/ml) in the presence or absence of TASQ (10µM). Subsequently, TLR4 downstreaming molecules such as IRAK1, gasdermin and NF-kB-p65 were analyzed by Western blot. Moreover, the mRNA expression of further proinflammatory molecules (IL-1b, IL-18, caspase1) and PD-L1 was quantified by real-time PCR. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with S100A9 ± TASQ before CD34+ hematopoietic stem and progenitor cells (HSPCs) were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly followed by a CFU-GEMM assay in methylcellulose medium. Additionally, PD-1 mRNA expression was quantified in cocultured HSPCs.ResultsImmunohistochemical staining of BM tissue demonstrated S100A9 expression mainly by CD66b+ neutrophils and with less extent by CD68+ macrophages. In line with this, we could not detect relevant S100A9 mRNA expression in cultured MDS or healthy MSCs in vitro.Exposure of MDS and healthy MSCs with S100A9 induced TLR4 downstream signalling as demonstrated by increased expression of IRAK1 and NF-kB-p65. We further detected a higher expression of gasdermin, an inductor of pyroptosis, in S100A9 exposed MSCs. Addition of TASQ abolished these effects and inhibited the expression of the mentioned proteins, indicating an alleviation of inflammation.Furthermore, we detected a 2-fold increase of mRNA expression of the proinflammatory cytokines IL-1b and IL-18 as well as a 5-fold increase of their activator caspase 1 in MSCs after treatment with S100A9, which could be prevented by TASQ. Interestingly, PD-L1 as a potential downstream target was induced by S100A9 by 2.5-fold and could be suppressed by TASQ to about 50%.To evaluate the impact on the hematopoietic support of MSCs, we analysed MSC/HSPC cocultures after treatment with S100A9. We observed a decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay, indicating a disturbed hematopoietic support by S100A9 treated MSCs. Interestingly, both the number of CAF-C and CFU could be increased by TASQ pre-treatment. Finally, the PD-1 expression in co-cultured HSPCs was regulated in the same way as its ligand in treated MSCs, nominating this interaction as a potential target of S100A9/TASQ in the MDS BM.ConclusionIn summary, we provide evidence that the pathological inflammasome activation in the myelodysplastic bone marrow can be rescued by TASQ at least in part by inhibition of the S100A9 mediated TLR4 downstream signalling including NF-kB-p65 transcription and PD-L1 expression. These effects result in an improved hematopoietic support by MSCs, suggesting a potential efficacy to improve cytopenia in low-risk MDS patients. DisclosuresBalaian: Novartis: Honoraria. Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. Platzbecker: AbbVie: Honoraria; Takeda: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; Janssen: Honoraria; Geron: Honoraria. Röllig: Novartis: Honoraria, Research Funding; Jazz: Honoraria; Janssen: Honoraria; Bristol-Meyer-Squibb: Honoraria, Research Funding; Amgen: Honoraria; AbbVie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Research Funding.

Tumor Microenvironment Uses a Reversible Reprogramming of Mesenchymal Stromal Cells to Mediate Pro-tumorigenic Effects.

The role of mesenchymal stromal cells (MSCs) in the tumor microenvironment is well described. Available data support that MSCs display anticancer activities, and that their reprogramming by cancer cells in the tumor microenvironment induces their switch toward pro-tumorigenic activities. Here we discuss the recent evidence of pro-tumorigenic effects of stromal cells, in particular (i) MSC support to cancer cells through the metabolic reprogramming necessary to maintain their malignant behavior and stemness, and (ii) MSC role in cancer cell immunosenescence and in the establishment and maintenance of immunosuppression in the tumor microenvironment. We also discuss the mechanisms of tumor microenvironment mediated reprogramming of MSCs, including the effects of hypoxia, tumor stiffness, cancer-promoting cells, and tumor extracellular matrix. Finally, we summarize the emerging strategies for reprogramming tumor MSCs to reactivate anticancer functions of these stromal cells.

Platelet-rich plasma-derived scaffolds increase the benefit of delayed mesenchymal stromal cell therapy after severe traumatic brain injury

BackgroundCell therapy using mesenchymal stromal cells (MSCs) offers new perspectives in the treatment of traumatic brain injury (TBI). The aim of the present study was to assess the impact of platelet-rich plasma scaffolds (PRPS) as support of MSCs in a delayed phase after severe TBI in rats. MethodsTBI was produced by weight-drop impact to the right cerebral hemisphere. Two months after TBI, four experimental groups were established; saline, PRPS, MSCs in saline, or MSCs in PRPS was transplanted into the area of brain lesion through a small hole. All groups were evaluated in the course of the following 12 months after therapy and the animals were then humanely killed. ResultsOur results showed that a greater functional improvement was obtained after the administration of MSCs in PRPS compared with the other experimental groups. DiscussionPRPS enhanced the benefit of cell therapy with MSCs to treat chronic brain damage in rats that suffered a severe TBI. The present findings suggest that the use of intralesional MSCs supported in PRPS may be a strategy of tissue engineering for patients with established neurological severe dysfunction after a TBI.

Regulation of mesenchymal stromal cells through fine tuning of canonical Wnt signaling

Mesenchymal stromal cells (MSCs) have been extensively utilized for various cell therapeutic trials, but the signals regulating their stromal function remain largely unclear. Here, we show that canonical Wnt signals distinctively regulate MSCs in a biphasic manner depending on signal intensity, i.e., MSCs exhibit proliferation and progenitor self-renewal under low Wnt/β-catenin signaling, whereas they exhibit enhanced osteogenic differentiation with priming to osteoblast-like lineages under high Wnt/β-catenin signaling. Moreover, low or high levels of β-catenin in MSCs distinctly regulated the hematopoietic support of MSCs to promote proliferation or the undifferentiated state of hematopoietic progenitors, respectively. A gene expression study demonstrated that different intracellular levels of β-catenin in MSCs induce distinct transcriptomic changes in subsets of genes belonging to different gene function categories. Different β-catenin levels also induced differences in intracellular levels of the β-catenin co-factors, Tcf-1 and Lef-1. Moreover, nano-scale mass spectrometry of proteins that co-precipitated with β-catenin revealed distinctive spectra of proteins selectively interacting with β-catenin at specific expression levels. Together, these results show that Wnt/β-catenin signals can coax distinct transcription milieu to induce different transcription profiles in MSCs depending on the signal intensity and that fine-tuning of the canonical Wnt signaling intensity can regulate the phase-specific functionality of MSCs.

Mesenchymal stromal cells support the viability and differentiation of follicular lymphoma-infiltrating follicular helper T-cells.

The biology of follicular lymphoma (FL) is largely dictated by the immune-effector and stromal cells that comprise its tumor microenvironment. FL-infiltrating T-cell populations that are thought to be fundamental to FL biology are follicular helper T-cells (TFH), follicular regulatory T-cells (TFR), a recently described population that regulates TFH activity, and regulatory T-cells (Treg). These T-cell populations have dynamic interactions with mesenchymal stromal cells (MSCs) in the tumor microenvironment. Whereas MSCs have been shown to support FL B-cell and Treg viability, their effects on FL-infiltrating TFH and TFR cells have not been described. Herein we show that MSCs support the viability of FL-infiltrating TFH and TFR, as well as Tregs, in part through an IL-6-dependent mechanism. We further demonstrate that MSCs mediate TFH to TFR conversion by inducing the expression of FoxP3 in TFH cells, demonstrating for the first time that human TFR can be derived from TFH cells. Given that the balance of TFH and TFR populations likely dictate, in part, the biology of this disease, our data support the potential for targeting MSCs as a therapeutic strategy.

Stromal Precursor Cells Characteristics In Patients With ALL Before and After Allogeneic Hematopoietic Stem Cell Transplantation

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow (BM). Lymphocytes connect tightly with stromal cells in BM. Leukemia and high dose chemotherapy affected both hematopoietic and stromal precursor cells. Changes in the hematopoiesis that occur during acute lymphoblastic leukemia (ALL) probably correlate with variation in the composition of stromal microenvironment.The aim of the study was to analyze the alterations occurring in stromal precursor cells - colony forming unit fibroblasts (CFU-F) and multipotent mesenchymal stromal cells (MSC) in patients with ALL before and after allogeneic hematopoietic stem cell transplantation (HSCT). HSCT following myeloblative conditioning was performed in 7 ALL patients (6 male, 1 female). After informed consent BM was aspirated before conditioning, and during the year after transplantation. MSC were cultured in aMEM with 10% fetal calf serum. Cumulative MSC production was counted after 5 passages. CFU-F was analyzed in standard conditions. The relative expression level (REL) of different genes was measured by RT-PCR. All data obtained at different time points were analyzed summarily. As a control MSC and CFU-F from 20 healthy donors of BM for HSCT were used after informed consent.The concentration of CFU-F in BM of ALL patients before HSCT was reduced by 12% in comparison with donors. After HSCT the concentration of CFU-F decreased further in 5.2 fold compared to the level before HSCT (p=0.02). Colonies formed by CFU-F depend on autocrine secretion of FGF2. It signals through 2 types of receptors, FGFR1 is considered to be the most important one. REL of FGFR1 was reduced by 27% in colonies from BM of patients before HSCT compared to donors. After HSCT it further decreased 1.2 fold. REL of FGFR2 in colonies of patients decreased 16 fold before and 50 fold after HSCT compared to donors (p=0.01 in both cases). REL of FGF2 in colonies of patients before HSCT doubled compared to donors and decreased 16 fold after HSCT. These data offer the molecular basis of decrease in CFU-F concentration in BM of patients after HSCT. In CFU-F colonies from patients REL of FGFR2 was decreased dramatically, however before HSCT it did not lead to decrease in CFU-F concentration due to increase in REL of FGF2. After HSCT REL of FGF2 and both receptors considerably decreased that led to significant reduction of CFU-F concentration in patients BM. REL of genes-markers of adipogenic and osteogenic differentiations were significantly decreased in CFU-F colonies of patients before HSCT (SPP1 in 8.6 fold, p=0.02, PPARG in 4.7 fold, p=0.01) pointing to less differentiated status of CFU-F progeny. After HSCT REL of these genes increased up to levels higher (PPARG in 3.5 fold, p=0.02) or comparable with donor ones, thus CFU-F seemed to regain “normal” differentiation status. REL of chondrogenic marker SOX9 did not change in CFU-F of ALL patients before and after HSCT, probably due to block of this differentiation lineage in CFU-F. REL of BMP4 in patients' CFU-F colonies was reduced 30 fold compared to donors (p=0.002), apparently due to damage made by leukemic сells. It increased 100 fold compared to donors after HSCT, that might reflect the intensive recovery of stromal microenvironment by interaction with non-malignant hematopoietic cells. Thus multiple alterations in CFU-F concentration and gene expression were revealed in CFU-F from BM of patients.MSC were also affected in ALL patients. Cumulative cell production in cultures from patients before HSCT was reduced by 35%, while after HSCT the decrease was more pronounced (by 61%, p=0.01). REL of genes regulating the proliferation (FGFR1, FGFR2 and FGF2) in patients MSC was halved in comparison to donors. After HSCT REL of both types of receptors did not change, while REL of FGF2 increased 1.5 fold not reaching donors' level. The decrease in cumulative cell production could depend on both FGF2 pathway alteration and on the increase of differentiation status in MSC population after HSCT. REL of differentiation markers in MSC before HSCT did not differ from donors' ones, while after HSCT REL of SPP1 increased 3.8 fold, PPARG – 2.3 fold (p=0.05) and SOX9 in 1.6 fold compared to donors. In patients MSC were less affected by leukemia and its treatment than CFU-F.This data suggest significant damage of stromal precursor cells in ALL patients that lasted at least for one year since HSCT. Disclosures:No relevant conflicts of interest to declare.

Mesenchymal Stromal Cells Support Follicular Lymphoma-Associated Follicular Helper T-Cells, in Part, Through an IL-6 and IL-21-Dependent Mechanism.

Abstract 2707The biology of follicular lymphoma (FL) is largely dictated by the immune-effector and stromal cells that comprise its tumor microenvironment. Recent studies have demonstrated that various infiltrating T-cell populations can be predictive of clinical outcome, with two such populations, follicular helper T-cells (TFH) and regulatory T-cells (Treg) likely playing critical roles in the biology of this disease. TFH are normally responsible for the activation and maintenance of germinal center B-cells while Tregs suppress effector T-cell priming and function as well as suppress normal B-cell proliferation and differentiation. There is also data to support the notion that both of these cells have direct and/or indirect effects on FL B-cells. These cells and other immune-effector cells within the FL microenvironment have dynamic interactions with mesenchymal stromal cells (MSC), multipotent cells residing in most adult tissues, which can be recruited by FL B-cells into the tumor microenvironment where they inhibit anti-lymphoma T-cell responses and FL B-cell apoptosis. We hypothesized that such MSC play a role in modulating TFH viability, similar to what has been previously reported for Treg, and herein we show such a novel role for MSC, that being the selective support of FL-derived TFH populations.To determine the effect of MSC on the viability of discrete T-cell populations infiltrating FL lymph nodes (FLN), un-separated single cell suspensions (SCS) from FLN (n=13) were cultured for 48 hours alone or with normal tonsillar-derived MSC. The fold change in the proportion of CD4 T-cells that are TFH (CXCR5+PD-1+Bcl-6+), Tregs (CD25+FoxP3+), TH1 (T-bet+), TH2 (GATA-3+) or TH17 (ROR-gamma-t+) cells in SCS incubated with MSC was compared to that of SCS incubated without MSC. A 2.3-fold increase in the percentage of TFH (p=0.006) cells and a 2-fold increase in the percentage of Tregs (p=0.007) were observed in the SCS cultured with MSC compared to those cultured without MSC. The effect of MSC in maintaining TFH and Tregs was selective, as in contrast, the proportion of T-cells expressing the canonical TH1, TH2 and TH17 transcription factors was not increased after culture with MSC. B-cells provide survival signals to TFH and stromal cells provide survival signals to B-cells, therefore we next determined whether the stromal support of TFH was mediated via B-cells. Both purified FL T-cells and B-cell-depleted FL SCS cultured on MSC showed a similar increase in TFH (and Treg) populations as seen with un-separated FL-B SCS, indicating that MSC support of TFH and Tregs was independent of B-cells. MSC only partially supported TFH and Treg populations in transwell experiments suggesting a role for both cell-cell contact and soluble factors. In this regard, IL-6 and IL-21 are known to be secreted by MSC and to signal through gp130 receptors on TFH to induce the transcription of Bcl-6, which is required for TFH differentiation. Blocking IL-6 and IL-21 decreased MSC support of TFH by 42% (p=0.021), while reducing MSC support of Tregs by only 17% (p=0.018) suggesting that IL-6 and IL-21 mediate, in part, the protective effect that MSC have on TFH but that other cytokines are likely to play a role in supporting Tregs. Finally, we have shown that FLN-derived MSC support both FL T-cells and normal lymph node T-cells to a similar extent as the tonsillar MSC used in these experiments. This provides support for the use of tonsillar MSC in these experiments as we were able to generate and expand these more consistently than MSC from FLN, which allowed us to use a consistent MSC product for all experiments.These findings therefore demonstrate a new role for MSC in FL, that being to support FL TFH in addition to Treg populations in the tumor microenvironment. MSCs have been shown to support FL B-cell viability and suppress anti-lymphoma T-cell responses. This finding that MSCs support TFH cells, a population that may provide survival signals to FL B-cells, further supports the potential of targeting MSC as a novel therapeutic strategy for patients with FL. Disclosures:No relevant conflicts of interest to declare.

Intercellular cytosolic transfer correlates with mesenchymal stromal cell rescue of umbilical cord blood cell viability during ex vivo expansion

Background aims. Mesenchymal stromal cells (MSC) have been observed to participate in tissue repair and to have growth-promoting effects on ex vivo co-culture with other stem cells. Methods. In order to evaluate the mechanism of MSC support on ex vivo cultures, we performed co-culture of MSC with umbilical cord blood (UCB) mononuclear cells (MNC) (UCB-MNC). Results. Significant enhancement in cell growth correlating with cell viability was noted with MSC co-culture (defined by double-negative staining for Annexin-V and 7-AAD; P<0.01). This was associated with significant enhancement of mitochondrial membrane potential (P<0.01). We postulated that intercellular transfer of cytosolic substances between MSC and UCB-MNC could be one mechanism mediating the support. Using MSC endogenously expressing green fluorescent protein (GFP) or labeled with quantum dots (QD), we performed co-culture of UCB-MNC with these MSC. Transfer of these GFP and QD was observed from MSC to UCB-MNC as early as 24 h post co-culture. Transwell experiments revealed that direct contact between MSC and UCB-MNC was necessary for both transfer and viability support. UCB-MNC tightly adherent to the MSC layer exhibited the most optimal transfer and rescue of cell viability. DNA analysis of the viable, GFP transfer-positive UCB-MNC ruled out MSC transdifferentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB-MNC viability (P< 0.0001). Conclusions. Collectively, the data suggest that intercellular transfer of cytosolic materials could be one novel mechanism for preventing UCB cell death in MSC co-culture.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271(bright)CD146(-) and CD271(bright)CD146(+) subsets correlates with donor age. The main subset in adults was CD271(bright)CD146(-), whereas the CD271(bright)CD146(+) population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271(-)CD146(+) cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions.

Co‐culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34+CD38− fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34+, CD133+ and CD38−) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.