Stephen E. Haynesworth

Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture‐expansion of these human marrow‐derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L‐ascorbic acid‐2‐phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM β‐glycerophosphate (βGP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti‐osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM βGP. The formation of a continuously interconnected network of APase‐positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number or APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end‐stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step‐wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes. J. Cell. Biochem. 64:295–312.

GROWTH, KINETICS, SELF-RENEWAL, AND THE OSTEOGENIC POTENTIAL OF PURIFIED HUMAN MESENCHYMAL STEM CELLS DURING EXTENSIVE SUBCULTIVATION AND FOLLOWING CRYOPRESERVATION

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self‐renewing capacity, and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle‐shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow‐derived adult human MSCs was determined to be 38 ± 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adults lifetime. J. Cell. Biochem. 64:278–294.

Characterization of cells with osteogenic potential from human marrow.

Studies using animal tissue suggest that bone marrow contains cells with the potential to differentiate into cartilage and bone. We report the extension of these studies to include human marrow. Bone marrow from male and female donors of various ages was obtained either from the femoral head or as aspirates from the iliac crest, and introduced into culture. Culture-adherent cells were expanded, subcultured, and then tested for bone and cartilage differentiation potential utilizing two different in vivo assays in nude mice. One assay involved subcutaneous implantation of porous calcium phosphate ceramics loaded with cultured, marrow-derived, mesenchymal cells; the other involved peritoneal implantation of diffusion chambers, also inoculated with cultured, marrow-derived, mesenchymal cells. Histological evaluation showed bone formation in ceramics implanted with cultured, marrow-derived, mesenchymal cells originating from both the femoral head and the iliac crest. Immunocytochemical analysis indicates that the bone is derived from the implanted human cells and not from the cells of the rodent host. No cartilage was observed in any of these ceramic grafts. In contrast, aliquots from the same preparations of cultured, marrow-derived, mesenchymal cells failed to form bone or cartilage in diffusion chambers. These data suggest that human marrow contains cells with osteogenic potential, which can be enriched and expanded in culture. Our findings also suggest that subcutaneous implantation of these cells in porous calcium phosphate ceramics may be a more sensitive in vivo assay than diffusion chambers for measuring their osteogenic lineage potential.

Rapid Hematopoietic Recovery After Coinfusion of Autologous-Blood Stem Cells and Culture-Expanded Marrow Mesenchymal Stem Cells in Advanced Breast Cancer Patients Receiving High-Dose Chemotherapy

PURPOSE Multipotential mesenchymal stem cells (MSCs) are found in human bone marrow and are shown to secrete hematopoietic cytokines and support hematopoietic progenitors in vitro. We hypothesized that infusion of autologous MSCs after myeloablative therapy would facilitate engraftment by hematopoietic stem cells, and we investigated the feasibility, safety, and hematopoietic effects of culture-expanded MSCs in breast cancer patients receiving autologous peripheral-blood progenitor-cell (PBPC) infusion. PATIENTS AND METHODS We developed an efficient method of isolating and culture-expanding a homogenous population of MSCs from a small marrow-aspirate sample obtained from 32 breast cancer patients. Twenty-eight patients were given high-dose chemotherapy and autologous PBPCs plus culture-expanded MSC infusion and daily granulocyte colony-stimulating factor. RESULTS Human MSCs were successfully isolated from a mean +/- SD of 23.4 +/- 5.9 mL of bone marrow aspirate from all patients. Expansion cultures generated greater than 1 x 10(6) MSCs/kg for all patients over 20 to 50 days with a mean potential of 5.6 to 36.3 x 10(6) MSCs/kg after two to six passages, respectively. Twenty-eight patients were infused with 1 to 2.2 x 10(6) expanded autologous MSCs/kg intravenously over 15 minutes. There were no toxicities related to the infusion of MSCs. Clonogenic MSCs were detected in venous blood up to 1 hour after infusion in 13 of 21 patients (62%). Median time to achieve a neutrophil count greater than 500/microL and platelet count >/= 20,000/microL untransfused was 8 days (range, 6 to 11 days) and 8.5 days (range, 4 to 19 days), respectively. CONCLUSION This report is the first describing infusion of autologous MSCs with therapeutic intent. We found that autologous MSC infusion at the time of PBPC transplantation is feasible and safe. The observed rapid hematopoietic recovery suggests that MSC infusion after myeloablative therapy may have a positive impact on hematopoiesis and should be tested in randomized trials.

Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies

Human bone marrow has been shown to contain mesenchymal cells, which fabricate the connective tissue network of the marrow called the stroma. A subset of these marrow-derived mesenchymal cells can be isolated, expanded in culture, and then induced to differentiate into bone-producing osteoblasts and ultimately osteocytes when placed in the proper environment. At present, there are no methods for definitively identifying these cells in human marrow tissue or following their differentiation into osteogenic phenotypes. Therefore, we culture-expanded, marrow-derived mesenchymal cells from human donors and used these cells to immunize cells from human donors and used these cells to immunize mice whose spleens were used to generate hybridoma cell lines, which secrete antibodies to antigens on the cell surface of these culture-expanded mesenchymal cells. Hybridoma culture supernatants were successively screened against highly enriched samples of culture-expanded, marrow-derived mesenchymal cells in cryosections and live cell cultures to identify unique cell surface antigens. Positive clones were then screened against cell suspensions of whole and fractionated marrow to identify hybridomas whose supernatants were nonreactive with marrow hemopoietic cells. Three hybridoma cell lines, SH2, SH3, and SH4, were identified; these hybridomas secrete antibodies that recognize antigens on the cell surface of marrow-derived mesenchymal cells, but fail to react with marrow-derived hemopoietic cells. Additional tissue screening reveals unique tissue distributions for each of the recognized antigens, which suggests different antigen recognition for each antibody. However, all three antibodies fail to react with the cell surface of osteoblasts or osteocytes, suggesting that the antigens recognized by these antibodies are developmentally regulated and specific for primitive or early-stage cells of the osteogenic lineage.

Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: Effects of dexamethasone and IL-1α

We previously reported the purification, culture‐expansion, and osteogenic differentiation potential of mesenchymal progenitor cells (MPCs) derived from human bone marrow. As a first step to establishing the phenotypic characteristics of MPCs, we reported on the identification of unique cell surface proteins which were detected with monoclonal antibodies. In this study, the phenotypic characterization of human marrow‐derived MPCs is further established through the identification of a cytokine expression profile under standardized growth medium conditions and in the presence of regulators of the osteogenic and stromal cell lineages, dexamethasone and interleukin‐1α (IL‐1α), respectively. Constitutively expressed cytokines in this growth phase include G‐CSF, SCF, LIF, M‐CSF, IL‐6, and IL‐11, while GM‐CSF, IL‐3, TGF‐β2, and OSM were not detected in the growth medium. Exposure of cells in growth medium to dexamethasone resulted in a decrease in the expression of LIF, IL‐6, and IL‐11. These cytokines have been reported to exert influence on the differentiation of cells derived from the bone marrow stroma through target cell receptors that utilize gp130‐associated signal transduction pathways. Dexamethasone had no effect on the other cytokines expressed under growth medium conditions and was not observed to increase the expression of any of the cytokines measured in this study. In contrast, IL‐1α increased the expression of G‐CSF, M‐CSF, LIF, IL‐6, and IL‐11 and induced the expression of GM‐CSF. IL‐1α had no effect on SCF expression and was not observed to decrease the production of any of the cytokines assayed. These data indicate that MPCs exhibit a distinct cytokine expression profile. We interpret this cytokine profile to suggest that MPCs serve specific supportive functions in the microenvironment of bone marrow. MPCs provide inductive and regulatory information which are consistent with the ability to support hematopoiesis, and also supply autocrine, paracrine, and juxtacrine factors that influence the cells of the marrow microenvironment itself. In addition, the cytokine profiles expressed by MPCs, in response to dexamethasone and IL‐1α, identify specific cytokines whose levels of expression change as MPCs differentiate or modulate their phenotype during osteogenic or stromagenic lineage entrance/progression.

Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation

Summary:Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and represent potential cellular therapy to enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease (GVHD). We investigated the role of human MSCs in NOD-SCID mice repopulation by unrelated human hematopoietic cells and studied the immune interactions between human MSCs and unrelated donor blood cells in vitro. When hematopoietic stem cell numbers were limited, human engraftment of NOD-SCID mice was observed only after coinfusion of unrelated human MSCs, but not with coinfusion of mouse mesenchymal cell line. Unrelated human MSCs did not elicit T-cell activation in vitro and suppressed T-cell activation by Tuberculin and unrelated allogeneic lymphocytes in a dose-dependent manner. Cell-free MSC culture supernatant, mouse stromal cells and human dermal fibroblasts did not elicit this effect. These preclinical data suggest that unrelated, human bone marrow-derived, culture-expanded MSCs may improve the outcome of allogeneic transplantation by promoting hematopoietic engraftment and limiting GVHD and their therapeutic potential should be tested in clinic.

Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages.

Human mesenchymal stem cells (MSCs), bone marrow-derived pluripotent adherent cells of mesenchymal origin can differentiate along the osteogenic, chondrogenic, adipogenic, and tendonogenic lineages. In this report we characterize cytokine and growth factor gene expression by MSCs and investigate the modulation of cytokine expression that occurs during osteogenic and stromal differentiation. MSCs constitutively expressed mRNA for interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), and stem cell factor (SCF). MSCs treated with IL-1alpha upregulated mRNA levels of IL-6, IL-11, and LIF, and began to express detectable levels of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF). mRNA levels of M-CSF and SCF did not change. MSCs cultured in osteogenic medium differentiated along the osteogenic lineage and downregulated mRNA levels of IL-6, IL-11 and LIF whereas, M-CSF and SCF expression were unchanged and G-CSF and GM-CSF remained undetectable. IL-3 was not detected in MSC culture under any conditions. MSCs precultured in control medium, IL-1alpha, or osteogenic medium maintained similar capacity to support long-term culture initiating cell (LT-CIC). Thus, primary and osteogenic differentiated MSCs produce important hematopoietic cytokines and support hematopoiesis in long-term cultures, suggesting that these cells may provide an excellent ex vivo environment for hematopoiesis during progenitor cell expansion and may be important for in vivo cell therapy.

Osteogenesis in marrow-derived mesenchymal cell porous ceramic composites transplanted subcutaneously: effect of fibronectin and laminin on cell retention and rate of osteogenic expression.

Cultured-expanded rat marrow-derived mesenchymal cells differentiate into osteoblasts when combined with a porous calcium phosphate delivery vehicle and subsequently implanted in vivo. In this study, the effects of ceramic pretreatment with the cell-binding proteins fibronectin and laminin on the osteogenic expression of marrow-derived mesenchymal cells were assessed by scanning electron microscopy, [3H]-thymidine-labeled cell quantitation, and histological evaluation of bone formation. Scanning electron microscopic observations showed that marrow-derived mesenchymal cells rapidly spread and attach to both fibronectin- or laminin-adsorbed ceramic surfaces but retain a rounded morphology on untreated ceramic surfaces. Quantitation of [3H]-thymidine labeled cells demonstrated that laminin and fibronectin preadsorbed ceramics retain approximately double the number of marrow-derived mesenchymal cells than do untreated ceramics harvested 1 wk postimplantation. Histological observations indicate that the amount of time required to first detect osteogenesis was shortened significantly by pretreatment of the ceramic with either fibronectin or laminin. Fibronectin- and laminin-coated ceramic composite samples were observed to contain bone within 2 wk postimplantation, while in untreated ceramic the earliest observation of bone was at 4 wk postimplantation. A comparison was made of the initial cell-loading, in vivo cell retention characteristics, and rate of osteogenesis initiation of marrow-derived mesenchymal cells on two types of ceramic with different pore structure and chemical composition, with and without preadsorption with fibronectin or laminin. “Biphasic” ceramics contain randomly distributed pores 200-400 μm in diameter, and “coral-based” ceramics have continuous pores of approximately 200 μm in diameter. Laminin or fibronectin preadsorption significantly increases the number of cells retained in all ceramic test groups by day 7 postimplantation. In addition, by day 7 postimplantation, the biphasic ceramics retain a significantly greater number of cells for all test groups than do coral-based ceramics. The biphasic ceramics consistently have more specimens positive for bone with the identical cell-loading conditions used throughout this study. These results indicate that the retention of cells within the ceramic is an important factor for optimization of marrow mesenchymal cell initiated bone formation. The retention of cells within ceramics is augmented by the adsorption of the cell-binding proteins laminin and fibronectin, but this effect varies depending on ceramic pore structure and/or chemical composition.

Human and animal mesenchymal progenitor cells from bone marrow: Identification of serum for optimal selection and proliferation

SummaryAn undifferentiated subset of cells within the stromal cell population of bone marrow in postnatal mammals retains the capacity to differentiate along osteogenic, adipogenic, fibroblastic, and chondrogenic lines. These cells, which are referred to as mesenchymal stem cells (MSCs), can be maintainedin vitro and expanded in number through a process of subculturing. MSCs are maintained in culture in medium supplemented with 10% fetal bovine serum (FBS). It is believed that certain, as yet unidentified, serum components play critical roles in the attachment and proliferation of MSCs. Commercially available FBS is poorly characterized and may vary in composition and quality from lot to lot. This study describes a method for the selection of lots of FBS that best support maintenance of the undifferentiated state, mitotic expansion of MSCsin vitro, and retention of multilineage developmental potential in response to appropriate cues.