Cotransplantation of CD34+ hematopoietic stem and progenitor cells (HSPCs) with mesenchymal stromal cells (MSCs) enhances HSPC engraftment. For these applications, MSCs are mostly obtained from bone marrow (BM). However, MSCs can also be isolated from the Wharton's jelly (WJ) of the human umbilical cord. This source, regarded to be a waste product, enables a relatively low-cost MSC acquisition without any burden to the donor. In this study, we evaluated the ability of WJ MSCs to enhance HSPC engraftment. First, we compared cultured human WJ MSCs with human BM-derived MSCs (BM MSCs) for in vitro marker expression, immunomodulatory capacity, and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSCs have a more restricted differentiation capacity, both WJ MSCs and BM MSCs expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Most importantly, cotransplantation of either WJ MSCs or BM MSCs with CB CD34+ cells into NOD SCID mice showed similar enhanced recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood, and spleen 6 weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon coincubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although stromal cell-derived factor-1 (SDF-1)-induced migration of CD34+ cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34+ cells were cultured on monolayers of WJ MSCs in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSCs replaced WJ MSCs in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34+ cell engraftment.
Ex vivo expansion of umbilical cord blood (UCB) is attempted to increase cell numbers to overcome the limitation of cell dose. Presently, suspension cultures or feeder mediated co-cultures are performed for expansion of hematopoietic stem cells (HSCs). Mesenchymal stem cells (MSCs) have proved to be efficient feeders for the maintenance of HSCs. Here, we have established MSCs-HSCs co-culture system with MSCs isolated from less invasive and ethically acceptable sources like umbilical cord tissue (C-MSCs) and placenta (P-MSCs). MSCs derived from these tissues are often compared with bone marrow derived MSCs (BM-MSCs) which are considered as a gold standard. However, so far none of the studies have directly compared C-MSCs with P-MSCs as feeders for ex vivo expansion of HSCs. Thus, we for the first time performed a systematic comparison of hematopoietic supportive capability of C and P-MSCs using paired samples.
UCB-derived CD34+ cells were isolated and co-cultured on irradiated C and P-MSCs for 10 days. C-MSCs and P-MSCs were isolated from the same donor. The cultures comprised of serum-free medium supplemented with 25 ng/ml each of SCF, TPO, Flt-3 L and IL-6. After 10 days cells were collected and analyzed for phenotype and functionality.
C-MSCs and P-MSCs were found to be morphologically and phenotypically similar but exhibited differential ability to support ex vivo hematopoiesis. Cells expanded on P-MSCs showed higher percentage of primitive cells (CD34+CD38−), CFU (Colony forming unit) content and LTC-IC (Long term culture initiating cells) ability. CD34+ cells expanded on P-MSCs also exhibited better in vitro adhesion to fibronectin and migration towards SDF-1α and enhanced NOD/SCID repopulation ability, as compared to those grown on C-MSCs. P-MSCs were found to be closer to BM-MSCs in their ability to expand HSCs. P-MSCs supported expansion of functionally superior HSCs by virtue of reduction in apoptosis of primitive HSCs, higher Wnt and Notch activity, HGF secretion and cell-cell contact. On the other hand, C-MSCs facilitated expansion of progenitors (CD34+CD38+) and differentiated (CD34−CD38+) cells by secretion of IL1-α, β, MCP-2, 3 and MIP-3α.
P-MSCs were found to be better feeders for ex vivo maintenance of primitive HSCs with higher engraftment potential than the cells expanded with C-MSCs as feeders.
Electronic supplementary material
The online version of this article (doi:10.1186/s13287-015-0194-y) contains supplementary material, which is available to authorized users.
Ex vivo HSC expansion; Cord mesenchymal stem cells; Placental mesenchymal stem cells
Studies have proposed that mesenchymal stem cells (MSCs) improve the hematopoietic engraftment in allogeneic or xenogeneic transplants and this is probably due to the MSCs’ immunosuppressive properties. Our study aimed to discern, for the first time, whether MSC infusion could facilitate the engraftment of hematopoietic stem cells (HSCs) in autologous transplantations models, where no immune rejection of donor HSCs is expected.
Recipient mice (CD45.2) mice, conditioned with moderate doses of radiation (5-7 Gy), were transplanted with low numbers of HSCs (CD45.1/CD45.2) either as a sole population or co-infused with increasing numbers of adipose-derived-MSCs (Ad-MSCs). The influence of Ad-MSC infusion on the short-term and long-term engraftment of donor HSCs was investigated. Additionally, homing assays and studies related with the administration route and with the Ad-MSC/HSC interaction were conducted.
Our data show that the co-infusion of Ad-MSCs with low numbers of purified HSCs significantly improves the short-term and long-term hematopoietic reconstitution of recipients conditioned with moderate irradiation doses. This effect was Ad-MSC dose-dependent and associated with an increased homing of transplanted HSCs in recipients’ bone marrow. In vivo and in vitro experiments also indicate that the Ad-MSC effects observed in this autologous transplant model are not due to paracrine effects but rather are related to Ad-MSC and HSC interactions, allowing us to propose that Ad-MSCs may act as HSC carriers, facilitating the migration and homing of the HSCs to recipient bone marrow niches.
Our results demonstrate that Ad-MSCs facilitate the engraftment of purified HSCs in an autologous mouse transplantation model, opening new perspectives in the application of Ad-MSCs in autologous transplants, including HSC gene therapy.
After cord blood (CB) transplantation, early platelet recovery in immune-deficient mice is obtained by expansion of CB CD34+ cells with thrombopoietin (TPO) as single growth factor. Moreover, improvement of hematopoietic engraftment has been shown by cotransplantation of mesenchymal stem cells (MSC). We investigated whether a combination of both approaches would further enhance the outcome of CB transplantation in NOD SCID mice. NOD SCID mice were transplanted with either CB CD34+ cells, CD34+ cells with MSC, TPO-expanded CD34+ cells or TPO-expanded CD34+ cells with MSC. We analyzed human platelet recovery in the peripheral blood (PB) from day 4 after transplantation onward and human bone marrow (BM) engraftment at week 6. The different transplants were assessed in vitro for their migration capacity and expression of CXCR4. TPO expansion improved the early platelet recovery in the PB of the mice. Cotransplantation of MSC with CD34+ cells improved BM engraftment and platelet levels in the PB 6 weeks after transplantation. Combining TPO expansion and MSC cotransplantation, however, neither resulted in a more efficient early platelet recovery, nor in a better BM engraftment, nor even very low or absent BM engraftment occurred. In vitro, MSC boosted the migration of CD34+ cells, suggesting a possible mechanism for the increase in engraftment. Our results show that cotransplantation of MSC with TPO-expanded CD34+ cells at most combines, but does not increase the separate advantages of these different strategies. A combination of both strategies even adds a risk of non engraftment.
Human mesenchymal stromal cells (MSC) have been shown to support the growth and differentiation of hematopoietic stem cells (HSC). We hypothesized that intra-osseous (IO) co-transplantation of MSC and umbilical cord blood (UCB) may be effective in improving early HSC engraftment, as IO transplantation has been demonstrated to enhance UCB engraftment in NOD SCID-gamma (NSG) mice. Following non-lethal irradiation (300rads), 6 groups of NSG mice were studied: 1) intravenous (IV) UCB CD34+ cells, 2) IV UCB CD34+ cells and MSC, 3) IO UCB CD34+ cells, 4) IO UCB CD34+ cells and IO MSC, 5) IO UCB CD34+ cells and IV MSC, and 6) IV UCB CD34+ and IO MSC. Analysis of human-derived CD45+, CD3+, and CD19+ cells 6 weeks following transplant revealed the highest level of engraftment in the IO UCB plus IO MSC cohort. Bone marrow analysis of human CD13 and CD14 markers revealed no significant difference between cohorts. We observed that IO MSC and UCB co-transplantation led to superior engraftment of CD45+, CD3+ and CD19+ lineage cells in the bone marrow at 6 weeks as compared with the IV UCB cohort controls. Our data suggests that IO co-transplantation of MSC and UCB facilitates human HSC engraftment in NSG mice.
cord blood stem cell transplantatio; mesenchymal stromal cells; hematopoietic stem cell transplantation
Inadequate engraftment of hematopoietic stem cells (HSCs) following in-utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to SDF1 ligand present in the bone marrow (BM) stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF1 axis.
We used the fetal sheep large animal model to test our hypotheses that: a) by administering plerixafor in-utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, b) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches, and c) by increasing the CXCR4+ fraction of CD34+ HSCs, we could improve engraftment. Human cord blood-derived CD34+ cells and human bone marrow-derived MSCs were used for these studies.
When MSCs were transplanted one week prior to CD34+ cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34+ cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4+ cells in the CD34+ population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages.
Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4-SDF1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.
hematopoietic stem cell transplantation; in utero transplantation; CXCR4; SDF1; plerixafor; sheep model
Multipotent mesenchymal stromal cells (MSC) are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments) when compared to adult MSC (2/10 experiments). However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age. These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.
Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Mesenchymal stem cells (MSCs) are traditionally isolated from marrow. Bone marrow–derived MSCs (BM-MSCs) have previously demonstrated their ability to act as a feeder layer in support of ex vivo cord blood expansion. However, the use of BM-MSCs to support the growth, differentiation, and engraftment of cord blood may not be ideal for transplant purposes. Therefore, the potential of MSCs from a novel source, the Wharton’s jelly of umbilical cords, to act as stromal support for the long-term culture of cord blood HSC was evaluated.
STUDY DESIGN AND METHODS
Umbilical cord–derived MSCs (UC-MSCs) were cultured from the Wharton’s jelly of umbilical cord segments. The UC-MSCs were then profiled for expression of 12 cell surface receptors and tested for their ability to support cord blood HSCs in a long-term culture-initiating cell (LTC-IC) assay.
Upon culture, UC-MSCs express a defined set of cell surface markers (CD29, CD44, CD73, CD90, CD105, CD166, and HLA-A) and lack other markers (CD45, CD34, CD38, CD117, and HLA-DR) similar to BM-MSCs. Like BM-MSCs, UC-MSCs effectively support the growth of CD34+ cord blood cells in LTC-IC assays.
These data suggest the potential therapeutic application of Wharton’s jelly–derived UC-MSCs to provide stromal support structure for the long-term culture of cord blood HSCs as well as the possibility of cotransplantation of genetically identical, HLA-matched, or unmatched cord blood HSCs and UC-MSCs in the setting of HSC transplantation.
Introduction: Mesenchymal stem cells (MSCs) contribute to the engraftment of transplanted hematopoietic stem cells (HSCs). MSCs also accelerate hematological recovery by secreting SDF-1 and enabling HSCs to enter the bone marrow (BM) via the SDF-1/CXCR4 axis. HOXB4 has been shown to stimulate HSC self-renewal. In this study, we examined whether SDF-1 and HOXB4 expression in MSCs co-transplanted with HSCs could synergistically improve hematopoietic recovery in irradiated mice. Methods: Using recombinant adenoviruses, we generated genetically modified BM-MSCs that expressed SDF-1, HOXB4, and an SDF-1/HOXB4 fusion gene. We then co-transplanted these modified MSCs with HSCs and investigated blood cell counts, BM cellularity, degree of human HSC engraftment, and survival rate in irradiated mice. Results: We found that co-culturing the SDF-1/HOXB4 fusion gene-modified MSCs (SDF-1/HOXB4-MSCs) and human umbilical cord blood CD34+ cells significantly improved HSC cell expansion in vitro. More importantly, co-transplantation of CD34+ cells and SDF-1/HOXB4-MSCs markedly increased the hematopoietic potential of irradiated mice as evidenced by the rapid recovery of WBC, PLT and HGB levels in peripheral blood and of BM cellularity. Co-transplantation also markedly improved engraftment of human CD45+ cells in mouse BM. Conclusions: Our study demonstrates that SDF-1/HOXB4-MSCs markedly accelerate hematopoietic recovery and significantly improve survival among mice treated with a lethal dose of irradiation. Therefore, SDF-1/HOXB4-MSCs could have therapeutic value by improving the efficacy of clinical transplantations in patients with defective hematopoiesis.
Mesenchymal stem cells; hematopoietic stem cells; SDF-1 gene; HOXB4 gene; irradiation; hematopoietic reconstitution; NOD/SCID mice
Mesenchymal stem cells (MSCs) have recently been identified and characterized in humans. Moreover, MSC secrete cytokines that can support hematopoietic progenitor growth. In the present study, we evaluated whether the efficacy of hematopoietic stem cell transplantation is improved by their co-transplantation with MSC, and whether this is positively correlated with the dose of infused MSCs. Accordingly, irradiated NOD/SCID mice were transplanted with 1×105 human CD34+ cells in the presence or absence of culture expanded MSCs (1×106 or 5×106). We evaluated human hematopoietic cell engraftment by flow cytometry and assessed MSC tissue distributions by fluorescence in situ hybridization. We found that CD45+ and CD34+ cell levels were significantly elevated in a dose-dependent manner in cotransplanted mice 4 weeks after transplantation. The engraftments of CD33+ and CD19+ cells also increased dose-dependently. However, the engraftment of CD3+ cells did not increase after co-transplantation with MSCs. Human Y chromosome+ cells were observed in multiple tissues and were more frequently observed in mice co-transplanted with 5×106 rather than 1×106 MSCs. These results suggest that MSCs are capable of enhancing hematopoietic cell engraftment and distribution in multiple organs in a dose-dependent fashion.
Mesenchymal Stem Cells; Hematopoietic Stem Cells; Transplantation
The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34+ progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (IV) or intrabone (IB) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.
Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.
We evaluated the effect of human parathyroid hormone (hPTH) on the engraftment and/or in vivo expansion of hematopoietic stem cells in an umbilical cord blood (UCB)-xenotransplantation model. In addition, we assessed its effect on the expression of cell adhesion molecules.
Materials and Methods
Female NOD/SCID mice received sublethal total body irradiation with a single dose of 250 cGy. Eighteen to 24 hours after irradiation, 1×107 human UCB-derived mononuclear cells (MNCs) and 5×106 human UCB-derived mesenchymal stem cells (MSCs) were infused via the tail vein. Mice were randomly divided into three groups: Group 1 mice received MNCs only, Group 2 received MNCs only and were then treated with hPTH, Group 3 mice received MNCs and MSCs, and were treated with hPTH.
Engraftment was achieved in all the mice. Bone marrow cellularity was approximately 20% in Group 1, but 70-80% in the hPTH treated groups. Transplantation of MNCs together with MSCs had no additional effect on bone marrow cellularity. However, the proportion of human CD13 and CD33 myeloid progenitor cells was higher in Group 3, while the proportion of human CD34 did not differ significantly between the three groups. The proportion of CXCR4 cells in Group 3 was larger than in Groups 1 and 2 but without statistical significance.
We have demonstrated a positive effect of hPTH on stem cell proliferation and a possible synergistic effect of MSCs and hPTH on the proportion of human hematopoietic progenitor cells, in a xenotransplantation model. Clinical trials of the use of hPTH after stem cell transplantation should be considered.
Umbilical cord blood; parathyroid hormone; bone marrow niches
Allogeneic hematopoietic stem cell transplantation for patients with a hemoglobinopathy can be curative but is limited by donor availability. Although positive results are frequently observed in those with an HLA-matched sibling donor, use of unrelated donors has been complicated by poor engraftment, excessive regimen-related toxicity, and graft-versus-host disease (GVHD). As a potential strategy to address these obstacles, a pilot study was designed that incorporated both a reduced-intensity conditioning and mesenchymal stromal cells (MSCs). Six patients were enrolled, including 4 with high-risk sickle cell disease (SCD) and 2 with transfusion-dependent thalassemia major. Conditioning consisted of fludarabine (150 mg/m2), melphalan (140 mg/m2), and alemtuzumab (60 mg for patients weighing > 30 kg and.9 mg/kg for patients weighing <30 kg). Two patients received HLA 7/8 allele matched bone marrow and 4 received 4-5/6 HLA matched umbilical cord blood as the source of HSCs. MSCs were of bone marrow origin and derived from a parent in 1 patient and from an unrelated third-party donor in the remaining 5 patients. GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. One patient had neutropenic graft failure, 2 had autologous hematopoietic recovery, and 3 had hematopoietic recovery with complete chimerism. The 2 SCD patients with autologous hematopoietic recovery are alive. The remaining 4 died either from opportunistic infection, GVHD, or intracranial hemorrhage. Although no infusion-related toxicity was seen, the cotransplantation of MSCs was not sufficient for reliable engraftment in patients with advanced hemoglobinopathy. Although poor engraftment has been observed in nearly all such trials to date in this patient population, there was no evidence to suggest that MSCs had any positive impact on engraftment. Because of the lack of improved engraftment and unacceptably high transplant-related mortality, the study was prematurely terminated. Further investigations into understanding the mechanisms of graft resistance and development of strategies to overcome this barrier are needed to move this field forward.
Hemoglobinopathies; Sickle cell disease; Thalassemia; Hematopoietic stem cell transplant; Reduced-intensity conditioning; Mesenchymal stromal cells; Umbilical cord; Bone marrow; Engraftment; Graft-versus-host disease
In a model of acute lung injury, embryonic stem cell-derived mesenchymal stem cells (ES-MSCs) were hypothesized to be more potent as a therapeutic agent than bone marrow-derived (BM)-MSCs owing to their more primitive source of origin. Both BM-MSCs and ES-MSCs markedly decreased endotoxin-induced inflammation. However, ES-MSCs did not show any beneficial effects in reducing pulmonary edema and lung protein permeability compared with BM-MSCs, suggesting that not all MSCs behave similarly.
Mesenchymal stem cells (MSCs) can be derived from multiple tissue sources. However, the optimal source of MSCs for cell-based therapy for acute lung injury (ALI) is unclear. In the present experiments, we studied bone marrow (BM)-derived and embryonic stem cell-derived human MSC (ES-MSCs) as a therapeutic agent in Escherichia coli endotoxin-induced ALI in mice. We hypothesized that ES-MSCs would be more potent than BM-MSCs owing to its more primitive source of origin. ALI was induced by the intratracheal instillation of endotoxin at 4 mg/kg into 10–12-week-old C57BL/6 mice with or without BM-MSCs, ES-MSCs, or normal human lung fibroblasts as a cellular control. Compared with the endotoxin-injured mice at 48 hours, the administration of ES-MSCs provided results similar to those of BM-MSCs, significantly reducing the influx of white blood cells and neutrophils and decreasing the secretion of the inflammatory cytokines, macrophage inflammatory protein-2 and tumor necrosis factor-α, in the injured alveolus. BM-MSCs also reduced extravascular lung water, a measure of pulmonary edema, by 60% and the total protein levels, a measure of lung permeability, by 66%. However, surprisingly, ES-MSCs did not have these protective effects, which was partially explained by the increased secretion of matrix metallopeptidase 9 by ES-MSCs, an enzyme known to increase lung protein permeability. In conclusion, both BM-MSCs and ES-MSCs markedly decreased endotoxin-induced inflammation. However, ES-MSCs did not show any beneficial effect on reducing pulmonary edema and lung protein permeability compared with BM-MSCs, suggesting that not all MSCs behave in a similar fashion. Our results highlight the need perhaps for a disease-specific potency assay for MSCs.
To determine the optimal source of mesenchymal stem cells (MSCs) for cell-based therapy for acute lung injury, bone marrow (BM)- and embryonic stem cell-derived human MSC (ES-MSCs) were compared as therapeutic agents for Escherichia coli endotoxin-induced lung injury in mice. ES-MSCs behaved similarly to BM-MSCs by markedly decreasing the inflammatory response induced by endotoxin. However, unlike BM-MSCs, ES-MSCs provided no protective effects against increasing lung water and protein permeability, in part because of an increase in expression of matrix metallopeptidase 9 by ES-MSCs. In patients with acute respiratory distress syndrome, impaired alveolar fluid clearance (i.e., no resolution of pulmonary edema fluid) has been associated with higher mortality rates. Although ES-MSCs might ultimately be found to have properties superior to those of BM-MSCs, such as for immunomodulation, these results highlight the need for a disease-specific potency assay for stem cell-based therapy.
Acute lung injury; Embryonic stem cells; Matrix metallopeptidase; Mesenchymal stem cell
The mechanism that regulates self-renewal and differentiation of hematopoietic stem cells (HSC) is a central question in stem cell biology that might ultimately lead to reliable protocols for in vitro expansion of HSC. Cellular fate is governed by cell-cell interaction with the microenvironment in the bone marrow, the stem cell niche. Mesenchymal stromal cells (MSC) are precursors of the cellular components, and they secrete extracellular matrix proteins of the bone marrow stroma. Therefore, MSC feeder layer might provide a suitable in vitro model system for the stem cell niche. In vitro assays demonstrate that MSC maintain the stem cell function of HSC and that MSC from bone marrow have a higher hematopoiesis supportive activity than MSC from adipose tissue. Co-cultivation with MSC might pave the way for expansion of long-term repopulating HSC, and various clinical trials indicate that co-transplantation of HSC and MSC might enhance engraftment. Thus, MSC are promising tools to elucidate the underlying mechanism of the cellular microenvironment. The large variety of preparative protocols for isolation and cultivation of MSC affects their stromal activity. Standardized isolation methods and molecular characterization of MSC are of utmost importance for reproducible isolation of hematopoiesis supportive stromal cells and for their potential clinical application.
Mesenchymal stem cell; Culture conditions; Microenvironment; Differentiation; Hematopoietic stem cells; Cell-cell interaction
Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme β-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.
Mesenchymal stem cells; Xenotransplantation; Ex vivo gene therapy; Adult stem cells; Lysosomal storage disease; Lentiviral vector
Rationale: Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases.
Objectives: We hypothesized that human umbilical cord blood stem cells might be an effective alternative to adult bone marrow and embryonic stem cells for regeneration and repair of injured airway epithelium.
Methods: Human cord blood was obtained from normal deliveries at the University of Vermont. Cultured plastic adherent cells were characterized as mesenchymal stem cells (MSCs) by flow cytometry and differentiation assays. Cord blood–derived MSCs (CB-MSCs) were cultured in specialized airway growth media or with specific growth factors, including keratinocyte growth factor and retinoic acid. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. CB-MSCs were systematically administered to immunotolerant, nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice. Lungs were analyzed for presence of human cells.
Measurements and Main Results: When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed Clara cell secretory protein (CCSP), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein C, and thyroid transcription factor-1 mRNA, and CCSP and CFTR protein. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression.
Conclusions: CB-MSCs appear to be comparable to MSCs obtained from adult bone marrow in ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo.
cord blood stem cells; lung epithelium; lung remodeling; mesenchymal stem cells
Mesenchymal stem cells (MSC) are multipotent in nature and believed to facilitate the engraftment of hematopoietic stem cells (HSC) when transplanted simultaneously in animal studies and even in human trials. In this study, we transfected culture-expanded MSC with granulocyte macrophage-colony stimulating factor (GM-CSF) and stem cell factor (SCF) cytokine genes and then cotransplanted with mononuclear cells (MNC) to further promote HSC engraftment. MNC were harvested from cord blood and seeded in long-term culture for ex vivo MSC expansion. A total of 1×107 MNC plus MSC/µL were introduced to the tail vein of nonobese diabetic/severe combined immunodeficiency mice. After 6-8 weeks later, homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization studies. The total nucleated cell count and the engraftment of CD45+/CD34+ cells and XX or XY positive human cells were significantly increased in cotransplanted mice and even higher with the cytokine gene-transfected MSC (GM-CSF>SCF, p<0.05) than in transplantation of MNC alone. These results suggest that MSC transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment. Delivering genes involved in homing and cell adhesions, CXCR4 or VLA, would further increase the efficiency of stem cell transplantation in the future.
Mesenchymal Stem Cell; Hematopoietic Stem Cell Transplantation; GM-CSF; Stem Cell Factor; Engraftment, Transfection; Cord Blood
We have developed NOD-Rag2null IL-2Rγnull (NR2G)
mice similar to NOD-scidIL-2Rγnull (NOG) mice
that are known as an excellent host to generate humanized mice. To evaluate the usefulness
of NR2G mice as a host for humanized mice, the engraftment rates and differentiation of
human cells after human hematopoietic stem cell (HSC) transplantation were compared among
NR2G, NOG, and NOD-scid mice. For this purpose, the appropriate
irradiation doses to expand the niche for human stem cells in the bone marrow were first
determined. As a result, 8 and 2.5 Gy in adult, and 4 and 1 Gy in newborn NR2G and NOG
mice, respectively, were found to be appropriate. Next, 5 × 104 human umbilical
cord blood CD34+ cells were intravenously inoculated into irradiated adult or
newborn of the immunodeficient mice. These HSC transplantation experiments demonstrated
that both NR2G and NOG mice showed high engraftment rates compared with
NOD-scid mice, although NOG mice showed a slightly higher engraftment
rate than that for NR2G mice. However, no difference was found in the human cell
populations differentiated from HSCs between NR2G and NOG mice. The HSC transplantation
experiments to adults and newborns of two immunodeficient mice also revealed that the HSC
transplantation into newborn mice resulted in higher engraftment rate than those into
adults. These results showed that NR2G mice could be used as an alternative host to NOG
mice to generate humanized mice.
humanized mice; immunodeficient mice; NOD-Rag2null IL-2Rγnull mice; NOG mice
Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34+ hematopoietic cells. Co-transplantation of CD34+ HSCs and CD34− MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromized mice yielded vascularized tissue. The average vascular number of co-transplanted CD34+ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34+ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34+ cells. Based on additional in vitro results of endothelial differentiation of CD34+ cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34+ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34+ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.
Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues, including bone marrow, adipose, and mucosa. MSCs have the capacity for self-renewal and differentiation. Reports have been published on the systemic administration of MSCs leading to functional improvements by engraftment and differentiation, thus providing a new strategy to regenerate damaged tissues. Recently, it has become clear that MSCs possess immunomodulatory properties and can therefore be used to treat diseases. However, the therapeutic effect mechanisms of MSCs are yet to be determined. Here, we investigated these mechanisms using a medication-related osteonecrosis of the jaw (MRONJ)-like mouse model.
To generate MRONJ-like characteristics, mice received intravenous zoledronate and dexamethasone two times a week. At 1 week after intravenous injection, maxillary first molars were extracted, and at 1 week after tooth extraction, MSCs were isolated from the bone marrow of the mice femurs and tibias. To compare “diseased MSCs” from MRONJ-like mice (d-MSCs) with “control MSCs” from untreated mice (c-MSCs), the isolated MSCs were analyzed by differentiation and colony-forming unit-fibroblast (CFU-F) assays and systemic transplantation of either d-MSCs or c-MSCs into MRONJ-like mice. Furthermore, we observed the exchange of cell contents among d-MSCs and c-MSCs during coculture with all combinations of each MSC type.
d-MSCs were inferior to c-MSCs in differentiation and CFU-F assays. Moreover, the d-MSC-treated group did not show earlier healing in MRONJ-like mice. In cocultures with any combination, MSC pairs formed cell–cell contacts and exchanged cell contents. Interestingly, the exchange among c-MSCs and d-MSCs was more frequently observed than other pairs, and d-MSCs were distinguishable from c-MSCs.
The interaction of c-MSCs and d-MSCs, including exchange of cell contents, contributes to the treatment potential of d-MSCs. This cellular behavior might be one therapeutic mechanism used by MSCs for MRONJ.
Mesenchymal stem cell; Medication-related osteonecrosis of the jaw; Therapeutic mechanism
AIM: To expand hematopoietic/progenitor stem cells (HS/PCs) from umbilical cord blood (UCB) and prepare the HS/PC product, and analyze preclinical transplantation and safety of HS/PC product.
METHODS: Human bone marrow-derived mesenchymal stem cells (MSCs) were used as feeder cells to expand HS/PCs from UCB in a serum-free culture system. The proliferation potential of HS/PCs was analyzed. The expanded HS/PCs were suspended in the L-15 medium to prepare the HS/PC product. The contamination of bacteria, fungi and mycoplasmas, the infection of exogenous virus, the concentration of bacterial endotoxin, and the SCF residual in HS/PC product were determined. Finally, cells from the HS/PC product with or without bone marrow-derived mesenchymal stem cells (BM-MSCs) were transplanted into the irradiated NOD/SCID mice to determine the in vivo engraftment potential.
RESULTS: After co-culture for 10 d, the total nuclear cells (TNCs) increased 125-fold, and CD34+ cells increased 43-fold. The granulocyte-macrophage colony- forming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) increased 3.3- and 4.7-fold respectively. The expanded cells were collected and prepared as the expanded product of HS/PCs by re-suspending cells in L-15 medium. For preclinical safety, the HS/PC product was analysed for contamination by bacteria, fungi and mycoplasmas, the bacterial endotoxin concentration and the SCF content. The results showed that the HS/PC product contained no bacteria, fungi or mycoplasmas. The bacterial endotoxin concentration was less than the detection limit of 6 EU/mL, and residual SCF was 75 pg/mL. Based on clinical safety, the HS/PC product was qualified for clinical transplantation. Finally, the HS/PC product was transplanted the irradiated mice where it resulted in rapid engraftment of hematopoietic cells.
CONCLUSION: HSPC product prepared from UCB in the serum-free culture system with hMSCs as feeder cells should be clinically safe and effective for clinical transplantation.
Hematopoietic stem cells; Ex vivo expansion; Preclinical safeties; Transplantation
Multipotent mesenchymal stem/stromal cells (MSCs) have been extensively used as a transplantable cell source for regenerative medicine and immunomodulatory therapy. Specifically in allogeneic hematopoietic stem cell transplantation (HSCT), co-transplantation or post-transplant infusion of MSCs derived from bone marrow (BM) of non-self donors has been implicated in accelerating hematopoietic recovery, ameliorating graft-vs.-host disease, and promoting tissue regeneration. However, irrespective of the use of MSC co-administration, post-transplant chimerism of BM-derived MSCs after allogeneic HSCT has been reported to remain of host origin, suggesting that the infused donor MSCs are immunologically rejected or not capable of long-term engraftment in the host microenvironment. Also, hematopoietic cell allografts currently used for HSCT do not seem to contain sufficient amount of MSCs or their precursors to reconstitute host BM microenvironment. Since the toxic conditioning employed in allo-HSCT may impair the function of host MSCs to maintain hematopoietic/regenerative stem cell niches and to provide a local immunomodulatory milieu, we propose that new directions for enhancing immunohematopoietic reconstitution and tissue repair after allogeneic HSCT include the development of strategies to support functional replenishment of residual host MSCs or to support more efficient engraftment of infused donor MSCs. Future areas of research should include in vivo tracking of infused MSCs and detection of their microchimeric presence in extra-marrow sites as well as in BM.
mesenchymal stem/stromal cells; hematopoietic cell transplantation; stem cell niches; hematopoietic reconstitution; graft-versus-host disease
Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)–rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into non-myelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis.
Myeloma; Bone disease; Cytotherapy; Osteoblasts; Placenta; Mesenchymal stem cells