Umbilical cord-derived mesenchymal stem cells promote myeloid-derived suppressor cell enrichment by secreting CXCL1 to prevent graft-versus-host disease after hematopoietic stem cell transplantation

Background aimsHuman mesenchymal stem cells (MSCs) from various tissues have emerged as attractive candidates for the prevention and treatment of graft-versus-host disease (GVHD). However, the molecular machinery that defines and channels the behavior of these cells remains poorly understood.MethodsIn this study, the authors compared the efficacy of four tissue-derived MSC types in controlling GVHD in a murine model and investigated their immunomodulatory effects.ResultsHuman umbilical cord-derived mesenchymal stem cells (hUCMSCs) effectively decreased the incidence and severity of GVHD, which was mediated by the enrichment of myeloid-derived suppressor cells in GVHD target tissues. RNA sequencing results showed that hUCMSCs highly expressed CXCL1.ConclusionsThese results suggest a novel prophylactic application of hUCMSCs for controlling GVHD after allogeneic hematopoietic stem cell transplantation.     

Human umbilical cord blood-derived stromal cells prevent graft-versus-host disease in mice following haplo-identical stem cell transplantation

Background aimsHuman umbilical cord blood-derived stromal cells (hUCBDSC) comprise a novel population of CD34⁺ cells that has been isolated in our laboratory. They have been shown previously not only to be non-immunogenic but also to exert immunosuppressive effects on xenogenic T cells in vitro. This study investigated the role of hUCBDSC in immunomodulation in an acute graft-versus-host disease (GvHD) mouse model after haplo-identical stem cell transplantationMethodsAcute GvHD was induced in recipient (B6 × BALB/c)F₁ mice by irradiation (750 cGy) followed by infusion of bone marrow cells and splenocytes from donor C57BL/6 mice. hUCBDSC were co-transplanted in the experimental group. The survival time, body weight and clinical and histopathologic scores were recorded after transplantation. The expression of surface markers [major histocompatibility complex (MHC) I, MHC II, CD80 and CD86] on CD11c⁺ dendritic cells (DC), and the percentage of CD4⁺ regulatory T cells (Treg), in the spleens of recipient mice were examined by flow cytometryResultsThe survival time was significantly prolonged, and the clinical and histopathologic scores were reduced in mice co-transplanted with hUCBDSC. The expression levels of the surface markers on DC were significantly lower in mice transplanted with hUCBDSC compared with those without. The proportion of CD4⁺ Treg in the spleen was also increased in mice transplanted with hUCBDSCConclusionsThese results from a GvHD mouse model are in agreement with previous in vitro findings, suggesting that hUCBDSC possess immunosuppressive properties and may act via influencing DC and CD4⁺ Treg.     

Human umbilical cord blood-derived stromal cells, a new resource in the suppression of acute graft-versus-host disease in haploidentical stem cell transplantation in sublethally irradiated mice

Human umbilical cord blood-derived stromal cells (hUCBDSCs), a novel population isolated from CD34(+) cells by our laboratory, exerted an immunosuppressive effect on xenogenic T cells. This study aimed to investigate whether hUCBDSCs play a critical role in the suppression of acute graft-versus-host disease (aGVHD). The hUCBDSCs were co-cultured with splenocytes (SPCs) of donor C57BL/6 mice. The aGVHD in the recipient (B6×BALB/c) F1 mice was induced by the infusion of bone marrow cells and SPCs from donor mice following sublethal irradiation. The shift in vivo for hUCBDSCs was detected. The proliferation and cell cycle of SPCs were tested by cell counting kit-8 and flow cytometry, respectively. The expression of CD49b natural killer (NK) cells and CD3 T cells was detected by flow cytometry in co-culture and post-transplantation. IL-4, and IFN-γ were detected by ELISA in the serum of co-culture and post-transplantation. The survival time, body weight, clinical score, and histopathological score were recorded for mice post-transplantation. The hUCBDSCs promoted the proliferation of SPCs and significantly increased the ratio of the S and G(2)/M phase (p < 0.05). The hUCBDSCs significantly increased the expression of CD49b NK cells and IL-4 protein and decreased the expression of CD3 T cells and IFN-γ protein both in vitro and in vivo. The survival time of mice with co-transplantation of hUCBDSCs was significantly prolonged, and decreased clinical and histopathological scores were also observed. The hUCBDSCs were continually detected in the target organs of GVHD. These results suggest that hUCBDSCs possess the capability of suppressing aGVHD, possibly via their influence on CD3 T cells, NK cells, and cytokines.     

Umbilical cord mesenchymal stem cells suppress B-cell proliferation and differentiation

Mesenchymal stem cells (MSCs) may be obtained from umbilical cord as an abundant and noninvasive source. However, the immunomodulatory properties of umbilical cord-MSCs (UC-MSCs) were poorly studied. In this study, we aimed to investigate the effects of UC-MSCs on B-cell proliferation and differentiation. UC-MSCs were found to suppress the proliferation of B cells isolated from murine spleen. Moreover, UC-MSCs markedly suppressed B-cell differentiation as shown by the decreased number of CD138+cells and reduced levels of IgM and IgG production in coculture. As revealed by transwell experiments, soluble factors produced by UC-MSCs might be involved in mediating B-cell suppression. The Blimp-1 mRNA expression was suppressed whereas the PAX-5 mRNA expression was induced in coculture. Finally, UC-MSCs modified the phosphorylation pattern of Akt and p38 pathways, which were involved in B-cell proliferation and differentiation. These results may further support the potential therapeutic use of UC-MSCs in treating autoimmune disorders.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

Effects of hypoxia on proliferation of human cord blood-derived mesenchymal stem cells

The purpose of our study was to examine the influence of hypoxia on proliferation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). The mononuclear cells were separated by density gradient centrifugation from human umbilical cord blood and then, respectively, cultured under hypoxia (5 % O₂) or normoxia (20 % O₂). Their cell morphology, cell surface markers, β-galactosidase staining, cell growth curve, DNA cycle, and the expression of hypoxia-inducible factor-1α (HIF-1α) were evaluated. We found that hypoxia, in part via HIF-1α, improved the proliferation efficiency, and prevented senescence of hUCB-MSCs without altering their morphology and surface markers. These results demonstrated that hypoxia provides a favorable culture condition to promote hUCB-MSCs proliferation in vitro, which is a better way to obtain sufficient numbers of hUCB-MSCs for research and certainly clinical application.     

Inflammatory cytokines and dendritic cells in acute graft-versus-host disease after allogeneic stem cell transplantation

The wider use of allogeneic stem cell transplantation (allo-SCT) is still limited by the immunologic recognition and destruction of host tissues, termed graft-versus-host disease (GVHD). The role of inflammatory cytokines such as TNF-alpha and IL-1, and their impact on immune effectors (mainly CD4+ and CD8+ T) cells has been extensively studied in the context of GVHD occurring after standard myeloablative allo-SCT. However, recent data suggested that GVHD pathophysiology is likely to involve more complex interactions where antigen-presenting cells, especially dendritic cells (DCs), may play a major role at time of initiation of acute GVHD. In addition, the wider use of reduced intensity and less toxic conditioning (RIC) regimens prior to allo-SCT would allow better visualization of the fine functions of immune effectors, thereby offering a window of opportunities to better decipher the intimate pathophysiological mechanisms underlying GVHD. The aim of this work is to review the available research evidence on the role of DCs as in vivo regulators of alloimmune reactivity, and their interactions with other immune effectors.     

Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues, with human umbilical cord (UC) providing an abundant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone marrow and cord blood. However, the mechanisms and cellular interactions used by MSCs to control immune responses remain to be fully elucidated. In this paper, we report that suppression of mitogen-induced T cell proliferation by human UC-, bone marrow-, and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly inhibited the ability of UC-MSCs to suppress T cell proliferation, indicating an important role for PGE(2). Monocytes purified from UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell proliferation assays, an effect mediated in part by UC-MSC PGE(2) production and enhanced by PBMNC alloactivation. Therefore, we identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell proliferation.     

Umbilical cord blood mesenchymal stem cells application in hematopoietic stem cells expansion on nanofiber three-dimensional scaffold

Umbilical cord blood (UCB) hematopoietic stem cells (HSCs) transplantation (HSCTs) is considered as a therapeutic strategy for malignant and nonmalignant hematologic disorders. Nevertheless, the low number of HSCs obtained from each unit of UCB can be a major challenge for using these cells in adults. In addition, UCB is a rich source of mesenchymal stem cells (MSCs) creating hopes for nonaggressive and painless treatment in tissue engineering compared with bone marrow MSCs. This study was designed to evaluate the effects of UCB-MSCs application in UCB-HSCs expansion on the nanoscaffold that mimics the cell's natural niche. To achieve this goal, after flow cytometry confirmation of isolated HSCs from UCB, they were expanded on three-dimensional (3D) poly-l -lactic acid (PLLA) scaffolds fabricated by electrospinning and two-dimensional (2D)-culture systems, such as (1) HSCs-MSCs culturing on the scaffold, (2) HSCs culturing on the scaffold, (3) HSCs-MSCs culturing on 2D, and (4) HSCs culturing on 2D. After 7 days, real-time polymerase chain reaction (PCR) was performed to evaluate the CXCR4 gene expression in the mentioned groups. Moreover, for the next validation, the number of total HSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, scanning electron microscopy imaging, and colony-forming unit assay were evaluated as well. The results of the study indicated that UCB-MSCs interaction with HSCs in 3D-culture systems led to the highest expansion of UCB-HSCs on day 7. Flow cytometry results showed the highest purity of HSCs cocultured with MSCs. Real-time PCR showed a significant increase in gene expression of CXCR4 in the mentioned group. The highest viability and clonogenicity were detected in the mentioned group too. Considered together, our results suggest that UCB-HSCs and MSCs coculturing on PLLA scaffold could provide a proper microenvironment that efficiently promotes UCB-HSCs expansion and UCB-MSCs can also be considered as a promising candidate for UCB-HSCTs.     

Application of human umbilical cord blood-derived mesenchymal stem cells in disease models

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are regarded as an alternative source of bone marrow-derived mesenchymal stem cells because collection of cord blood is less invasive than that of bone marrow. hUCB-MSCs have recently been studied for evaluation of their potential as a source of cell therapy. In this review, the general characteristics of hUCB-MSCs and their therapeutic effects on various diseases in vitro and in vivo will be discussed.     

Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells attenuates Escherichia coli-induced acute lung injury in mice

Background

Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuate hyperoxic neonatal lung injury primarily through anti-inflammatory effects. We hypothesized that intratracheal transplantation of human UCB-derived MSCs could attenuate Escherichia coli (E. coli)-induced acute lung injury (ALI) in mice by suppressing the inflammatory response.

Methods

Eight-week-old male ICR mice were randomized to control or ALI groups. ALI was induced by intratracheal E. coli instillation. Three-hours after E. coli instillation, MSCs, fibroblasts or phosphate-buffered saline were intratracheally administered randomly and survival was analyzed for 7 days post-injury. Lung histology including injury scores, myeloperoxidase (MPO) activity, and protein levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 as well as the wet-dry lung ratio and bacterial counts from blood and bronchoalveolar lavage (BAL) were evaluated at 1, 3, and 7 days post-injury. Levels of inflammatory cytokines in the lung were also profiled using protein macroarrays at day 3 post-injury which showed peak inflammation.

Results

MSC transplantation increased survival and attenuated lung injuries in ALI mice, as evidenced by decreased injury scores on day 3 post-injury and reduced lung inflammation including increased MPO activity and protein levels of IL-1α, IL-1β, IL-6, TNF-α, and MIP-2 on day 3 and 7 post-injury. Inflammatory cytokine profiles in the lungs at day 3 post-injury were attenuated by MSC transplantation. MSCs also reduced the elevated lung water content at day 3 post-injury and bacterial counts in blood and BAL on day 7 post-injury.

Conclusions

Intratracheal transplantation of UCB-derived MSCs attenuates E. coli-induced ALI primarily by down-modulating the inflammatory process and enhancing bacterial clearance.     

Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation.

This paper reviews our recent laboratory and clinical studies demonstrating the efficacious use of human umbilical cord blood for HLA-matched allogeneic sibling stem/progenitor cell transplantation in cases of Fanconi's anemia. Future implications and potential problems are discussed with regards to (a) the possibility of maternal cell contamination, (b) the broadness of applicability with regards to other diseases that might be transplanted, and whether such transplants are feasible in adults, as well as in children, and (c) the immunological reactivity of cord blood cells, and whether these cells can be used to cross histocompatibility barriers more easily than that of bone marrow from adults.     

间充质干细胞在造血干细胞移植中的应用

间充质干细胞(MSCs)是一种具有自我更新和多向分化潜能的成体干细胞,存在于骨髓、脂肪组织、脐血及多种胎儿组织.它可分泌多种细胞因子及生长因子,促进造血干细胞(HSC)的增殖与分化.MSCs还具有免疫调节、抗炎和组织修复作用,可减轻移植物抗宿主病(GVHD)及其他移植相关并发症.     

Harnessing and honing mesenchymal stem/stromal cells for the amelioration of graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation is a deterministic curative procedure for various hematologic disorders and congenital immunodeficiency. Despite its increased use, the mortality rate for patients undergoing this procedure remains high, mainly due to the perceived risk of exacerbating graft-versus-host disease (GVHD). However, even with immunosuppressive agents, some patients still develop GVHD. Advanced mesenchymal stem/stromal cell (MSC) strategies have been proposed to achieve better therapeutic outcomes, given their immunosuppressive potential. However, the efficacy and trial designs have varied among the studies, and some research findings appear contradictory due to the challenges in characterizing the in vivo effects of MSCs. This review aims to provide real insights into this clinical entity, emphasizing diagnostic, and therapeutic considerations and generating pathophysiology hypotheses to identify research avenues. The indications and timing for the clinical application of MSCs are still subject to debate.     

Extracorporeal photopheresis as first-line strategy in the treatment of acute graft-versus-host disease after hematopoietic stem cell transplantation: A single-center experience

Background aims: Corticosteroids are the standard first-line treatment for acute graft-versus-host disease (aGVHD), but they are associated with many complications, and less than half of patients have a sustained response.Methods: To improve outcomes, we performed a retrospective study to analyze the efficacy of the addition of extracorporeal photopheresis (ECP) to low-dose corticosteroids in 37 adult patients (median age, 57 years) with skin-predominant aGVHD (grade I, n = 17; grade II, n = 18; and grade III, n = 2). All patients received ECP in combination with 1 mg/kg prednisone (n = 26) or topical steroids (n = 11).Results: Overall response rate was 81% after a median of three ECP procedures (range, 2–8), including 22 complete responses (CR, 59%) and eight very good partial responses (VGPR, 22%). The 11 patients treated with topical corticosteroids achieved CR. Furthermore, 16 (62%) patients reached prednisone withdrawal at a median of 100 days (range, 42–174 days) after its initiation. Eighteen patients developed chronic GVHD (cGVHD); 11 of them (who were in CR of aGVHD) had a new-onset cGVHD, and seven experienced progressive cGVHD (five non-responding and two VGPR patients). A second-line immunosuppressive treatment was initiated in only five (14%) non-responding patients. With a median follow-up of 31 months (range, 6–57 months) 2-year overall survival and non-relapse mortality were 74% and 11%, respectively.Conclusions: Overall, the combination of low-dose corticosteroids and ECP appear to be safe and effective for first-line treatment of skin predominant aGVHD.     

CD1d is a novel cell-surface marker for human monocytic myeloid-derived suppressor cells with T cell suppression activity in peripheral blood after allogeneic hematopoietic stem cell transplantation

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer and various pathological conditions. However, the phenotypic and functional heterogeneity of human MDSCs represents a major hurdle for the development of therapeutic strategies targeting or regulating MDSCs in tumor progression, inflammation, and graft-versus-host disease (GVHD). We previously shown that circulating HLA-DR^-CD14⁺ monocytic MDSCs are a major contributor to clinical outcomes after allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this study, we identified, using high-throughput screening, a set of surface markers that are strongly expressed in HLA-DR^-CD14⁺ monocytic MDSCs isolated from the peripheral blood (PB) of patients receiving allo-HSCT. Subsequent experiments showed the consistent dominant expression of CD1d in monocytic MDSCs of allo-HSCT PB in comparison with granulocytic MDSCs. In addition, CD1d-expressing cells isolated from PB of allo-HSCT patients showed the suppressive activity of T cell proliferation and higher expression of MyD88 and IDO compared with CD1d^? cells. Our results suggest that CD1d could be a valuable marker for further therapeutic evaluation of human monocytic MDSCs for immune-related diseases, including GVHD.     

Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion

The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increasedCD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.     

Human cord blood-derived viral pathogens as the potential threats to the hematopoietic stem cell transplantation safety:A mini review

Umbilical cord blood(UCB) is a valuable source of hematopoietic stem cells(HSCs) and potential alternative for bone marrow transplantation for patients who lack human leukocyte antigen(HLA)-matched donors. The main practical advantages of UCB over other HSC sources are the immediate availability, lower incidence of graft-versus-host disease, minimal risk to the donor, and lower requirement for HLA compatibility. However, the use of UCB is limited by delayed engraftment and poor immune reconstitution, leading to a high rate of infection-related mortality. Therefore, severe infectious complications, especially due to viral pathogens remain the leading cause of morbidity and mortality during the post-UCB transplantation(UCBT) period. In this context, careful screening and excluding the viral-contaminated UCB units might be an effective policy to reduce the rate of UCBT-related infection and mortality. Taken together,complete prevention of the transmission of donor-derived viral pathogens in stem cell transplantation is not possible. However, having the knowledge of the transmission route and prevalence of viruses will improve the safety of transplantation. To the best of our knowledge, there are few studies that focused on the risk of virus transmission through the UCB transplant compared to other HSC sources. This review summarizes the general aspects concerning the prevalence, characteristics, and risk factors of viral infections with a focus on the impact of viral pathogens on cord blood transplantation safety.     

Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix.