Role of mesenchymal stem cells in hematopoietic stem cell transplantation

Within the bone marrow stroma are multipotential cells which are capable of differentiation into a number of mesenchymal cell lineages. These cells, termed mesenchymal stem cells, have recently been identified and characterized in humans. Many studies indicate that the bone marrow stroma is damaged following bone marrow transplantation. Since the marrow stroma is critical for the maintenance of hematopoiesis, its ability to support hematopoiesis following stem cell transplantation may be impaired. Animal models suggest that the transplantation of healthy stromal elements, including mesenchymal stem cells, may enhance the ability of the bone marrow microenvironment to support hematopoiesis after stem cell transplantation. Here the authors review recent data that suggest that mesenchymal stem cells may possess therapeutic value not only for the repair of damaged mesenchymal tissues following hematopoietic stem cell transplantation, but also as potential vectors for the delivery of corrective genes.     

Regulation of hematopoiesis in endosteal microenvironments

After birth, the hematopoietic system develops along with bone formation in mammals. Osteolineage cells are derived from mesenchymal progenitor cells, and differentiate into several types of bone-forming cells. Of the various types of cell constituents in bone marrow, osteolineage cells have been shown to play important roles in hematopoiesis. Early studies have identified osteoblasts as a hematopoietic stem cell niche component. Since that time, the role of endosteal microenvironment as a critical regulator of hematopoietic stem/progenitor cell (HSC/HPC) behavior has been appreciated particularly under stress conditions, such as cytokine-induced HSC/HPC mobilization, homing/engraftment after bone marrow transplantation, and disease models of leukemia/myelodysplasia. Recent studies revealed that the most differentiated osteolineage cells, i.e., osteocytes, play important roles in the regulation of hematopoiesis. In this review, we provide an overview of recent advances in knowledge of regulatory hematopoietic mechanisms in the endosteal area.     

Mesenchymal stem cell: use and perspectives

Studies on hematopoiesis have focused on the function and composition of human bone marrow stroma. Stroma function gives hematopoietic stem cells the microenvironment appropriate for self-renewal and/or prompt differentiation into hematopoietic progenitor cells, then into terminal specialized cells. Human bone marrow stroma has been dissected into hematopoietic and nonhematopoietic components. The former includes hematopoietic-derived cells, mainly macrophages, while the latter, still poorly characterized, is composed mainly of endothelial and mesenchymal stem cells and their derivatives (adipocytes, chondrocytes, cells of the osteogenic lineage). Isolation of bone marrow mesenchymal stem cells has made available a population of adherent cells, belonging to the non-hematopoietic stroma, which are morphologically and phenotypically homogeneous. This review will focus on: (i) definition of bone marrow stroma and mesenchymal stem cells; (ii) methods of mesenchymal stem cell isolation, morphological and phenotypic characterization; (iii) mesenchymal stem cell functional and differentiation properties and (iv) therapeutic applications of mesenchymal stem cells.     

Hematopoiesis is severely altered in mice with an induced osteoblast deficiency

We previously reported a transgenic mouse model expressing herpesvirus thymidine kinase (TK) gene under the control of a 2.3-kilobase fragment of the rat collagen alpha1 type I promoter (Col2.3 Delta TK). This construct confers lineage-specific expression in developing osteoblasts, allowing the conditional ablation of osteoblast lineage after treatment with ganciclovir (GCV). After GCV treatment these mice have profound alterations on bone formation leading to a progressive bone loss. In addition, treated animals also lose bone marrow cellularity. In this report we characterized hematopoietic parameters in GCV-treated Col2.3 Delta TK mice, and we show that after treatment transgenic animals lose lymphoid, erythroid, and myeloid progenitors in the bone marrow, followed by decreases in the number of hematopoietic stem cells (HSCs). Together with the decrease in bone marrow hematopoiesis, active extramedullary hematopoiesis was observed in the spleen and liver, as measured by an increase in peripheral HSCs and active primary in vitro hematopoiesis. After withdrawal of GCV, osteoblasts reappeared in the bone compartment together with a recovery of medullary and decrease in extramedullary hematopoiesis. These observations directly demonstrate the role of osteoblasts in hematopoiesis and provide a model to study the interactions between the mesenchymal and hematopoietic compartments in the marrow.     

Human bone marrow stromal cell: coexpression of markers specific for multiple mesenchymal cell lineages

The role of hematopoietic stem cells in blood cell development is reasonably understood, whereas the identity and the function of bone marrow stromal cells are much less clear. Using stromal cells in bone marrow cultures of the Dexter type, a favorite medium for the study of hematopoiesis, we show that stromal cells actually represent a unique cell type. Conventional wisdom has held that stromal cells in Dexter cultures comprise a mixture of macrophages, hematopoietic cells, adipocytes, osteoblasts, fibroblasts, muscle cells, and endothelial cells. Our findings demonstrate that Dexter cultures consist of three cell types: macrophages ( approximately 35%), hematopoietic cells ( approximately 5%), and nonhematopoietic cells ( approximately 60%). We have purified the nonhematopoietic cells free of macrophages and hematopoietic cells to produce compelling evidence that they in fact represent a single cell type (multidifferentiated mesenchymal progenitor cell, MPC) which coexpresses genes specific for various mesenchymal cell lineages including adipocytes, osteoblasts, fibroblasts, and muscle cells. We further show that these multi- or pluridifferentiated MPCs are capable of supporting hematopoiesis by demonstrating the expression of several hematopoietic growth factors and extracellular matrix receptors including G-CSF, SCF, VCAM-1, ICAM-1, and ALCAM. Since the MPCs can be easily purified to near homogeneity (95%), they can be of value in enhancing engraftment of hematopoietic stem cells. Also, this new understanding of bone marrow stromal cells as "one cell with many different faces" promises to advance our knowledge of regulatory cellular interactions within bone marrow.     

Bone marrow cell death and proliferation: Controlling mechanisms in normal and leukemic state

Bone marrow cell death and proliferation are regulated by multiple factors including genetic and epigenetic alterations of hematopoietic cells, crosstalk of hematopoietic cells with bone marrow mesenchymal cells through direct cell-cell interaction or cytokine/chemokine production, vascularity of the bone marrow, and interactions of sympathetic nerve system with hematopoiesis. Cell proliferation usually predominates over cell death in neoplastic processes such as leukemia and myeloproliferative neoplasms, while apoptotic processes also have a significant role in the pathogenesis of myelodysplastic syndromes. Recently, hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) have been identified and their characters on self renewal process, differentiation, cell dynamics and drug resistance have been implicated. Although most leukemia cells are initially sensitive to chemo- or radiotherapy, LSCs are resistant and considered to be the basis for disease relapse after initial response. HSCs and LSCs may use similar interactions with bone marrow microenvironment. However, bone marrow microenvironment called niche should influence the normal as well as malignant hematopoiesis in different manners. Recent studies have expanded the number of cell types constituting bone marrow niche and made the issue more complex. Since the majority of excellent and contributing studies on bone marrow niches have been performed in animal models, niches in human tissues are beginning to be localized and characterized. In this article, we summarize the relation of hematopoietic cells with niches and hope to point a hint to the novel strategy for treatment of malignant proliferation of hematopoietic cells.     

SSEA-4 identifies mesenchymal stem cells from bone marrow

Adult bone marrow (BM) contains hematopoietic stem cells (HSCs) as well as a nonhematopoietic, stromal cell population. Within this stromal population are mesenchymal stem cells (MSCs), which not only support hematopoiesis but also differentiate into multiple lineages, including fat, bone, and cartilage. Because of this multipotentiality, the MSC is an attractive candidate for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, research progress has been hampered by the limited existing knowledge of the biology of these cells, particularly by the lack of a suitable marker for their prospective isolation. Here, we report that SSEA-4, an early embryonic glycolipid antigen commonly used as a marker for undifferentiated pluripotent human embryonic stem cells and cleavage to blastocyst stage embryos, also identifies the adult mesenchymal stem cell population.     

Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease

Aplastic anemia is a life-threatening bone marrow failure disorder characterized by peripheral pancytopenia and marrow hypoplasia. The majority of cases of aplastic anemia remain idiopathic, although hematopoietic stem cell deficiency and impaired immune responses are hallmarks underlying the bone marrow failure in this condition. Mesenchymal stem/stromal cells constitute an essential component of the bone marrow hematopoietic microenvironment because of their immunomodulatory properties and their ability to support hematopoiesis, and they have been involved in the pathogenesis of several hematologic malignancies. We investigated whether bone marrow mesenchymal stem cells contribute, directly or indirectly, to the pathogenesis of aplastic anemia. We found that mesenchymal stem cell cultures can be established from the bone marrow of aplastic anemia patients and display the same phenotype and differentiation potential as their counterparts from normal bone marrow. Mesenchymal stem cells from aplastic anemia patients support the in vitro homeostasis and the in vivo repopulating function of CD34⁺ cells, and maintain their immunosuppressive and anti-inflammatory properties. These data demonstrate that bone marrow mesenchymal stem cells from patients with aplastic anemia do not have impaired functional and immunological properties, suggesting that they do not contribute to the pathogenesis of the disease.     

Mesenchymal stem cells: biology and potential clinical uses

There has been an increasing interest in recent years in the stromal cell system functioning in the support of hematopoiesis. The stromal cell system has been proposed to consist of marrow mesenchymal stem cells that are capable of self-renewal and differentiation into various connective tissue lineages. Recent efforts demonstrated that the multiple mesenchymal lineages can be clonally derived from a single mesenchymal stem cell, supporting the proposed paradigm. Dexter demonstrated in 1982 that an adherent stromal-like culture was able to support maintenance of hematopoietic stem as well as early B lymphopoeisis. Recent data from in vitro models demonstrating the essential role of stromal support in hematopoiesis shaped the view that cell-cell interactions in the marrow microenvironment are critical for normal hematopoietic function and differentiation. Maintenance of the hematopoietic stem cell population has been used to increase the efficiency of hematopoietic stem cell gene transfer. High-dose chemotherapy and frequently cause stromal damage with resulting hematopoietic defects. Data from preclinical transplantation studies suggested that stromal cell infusions not only prevent the occurrence of graft failure, but they have an immunomodulatory effect. Preclinical and early clinical safety studies are paving the way for further applications of mesenchymal stem cells in the field of transplantation with respect to hematopoietic support, immunoregulation, and graft facilitation.     

Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment

The interactions between hematopoietic cells and the bone marrow (BM) microenvironment play a critical role in normal and malignant hematopoiesis and drug resistance. These interactions within the BM niche are unique and could be important for developing new therapies. Here, we describe the development of extramedullary bone and bone marrow using human mesenchymal stromal cells and endothelial colony-forming cells implanted subcutaneously into immunodeficient mice. We demonstrate the engraftment of human normal and leukemic cells engraft into the human extramedullary bone marrow. When normal hematopoietic cells are engrafted into the model, only discrete areas of the BM are hypoxic, whereas leukemia engraftment results in widespread severe hypoxia, just as recently reported by us in human leukemias. Importantly, the hematopoietic cell engraftment could be altered by genetical manipulation of the bone marrow microenvironment: Extramedullary bone marrow in which hypoxia-inducible factor 1α was knocked down in mesenchymal stromal cells by lentiviral transfer of short hairpin RNA showed significant reduction (50% ± 6%; P = .0006) in human leukemic cell engraftment. These results highlight the potential of a novel in vivo model of human BM microenvironment that can be genetically modified. The model could be useful for the study of leukemia biology and for the development of novel therapeutic modalities aimed at modifying the hematopoietic microenvironment.     

Zebrafish stromal cells have endothelial properties and support hematopoietic cells

The goal of this study was to determine if we could establish a mesenchymal stromal line from zebrafish that would support hematopoietic cells. Such a coculture system would be a great benefit to study of the hematopoietic cell-stromal cell interaction in both in?vitro and in?vivo environments. Zebrafish stromal cells (ZStrC) were isolated from the "mesenchymal" tissue of the caudal tail and expanded in a specialized growth media. ZStrC were evaluated for phenotype, gene expression, and ability to maintain zebrafish marrow cells in coculture experiments. ZStrC showed mesenchymal and endothelial gene expression. Although ZStrC lacked the ability to differentiate into classic mesenchymal stromal cell lineages (i.e., osteocytes, adipocytes, chondrocytes), they did have the capacity for endotube formation on Matrigel and low-density lipoprotein uptake. ZStrC supported marrow cells for >2 weeks in?vitro. Importantly, marrow cells were shown to retain homing ability in adoptive transfer experiments. ZStrC were also shown to improve hematopoietic recovery after sublethal irradiation after adoptive transfer. As the zebrafish model grows in popularity and importance in the study of hematopoiesis, new tools to aid in our understanding of the hematopoietic cell-stromal cell interaction are required. ZStrC represent an additional tool in the study of hematopoiesis and will be useful in understanding the factors that mediate the stromal cell-hematopoietic cell interactions that are important in hematopoietic cell maintenance.     

Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells?

In adult bone marrow, hematopoietic stem cells are found in close association with distinctive stromal cell elements. This association is necessary for maintenance of hematopoiesis, but the precise mechanisms underlying the cross-talk between stromal cells and hematopoietic stem cells are poorly understood. In this study, we used a bone marrow stromal cell line (MS-5) that is able to support human long-term hematopoiesis. This hematopoietic-promoting activity cannot be related to expression of known cytokines and is abolished by addition of hydrocortisone. Using a gene trap strategy that selects genes encoding transmembrane or secreted proteins expressed by MS-5 cells, we obtained several insertions that produced fusion proteins. In one clone, fusion protein activity was downregulated in the presence of hydrocortisone, and we show that insertion of the trap vector has occurred into the neuropilin-1 gene. Neuropilin-1 is expressed in MS-5 cells, in other hematopoietic-supporting cell lines, and in primary stromal cells but not in primitive hematopoietic cells. We show that neuropilin-1 acts as a functional cell-surface receptor in MS-5 cells. Two neuropilin-1 ligands, semaphorin III and VEGF 165, can bind to these cells, and the addition of VEGF 165 to MS-5 cells increases expression of 2 cytokines known to regulate early hematopoiesis, Tpo and Flt3-L. Finally, we show that stromal cells and immature hematopoietic cells express different neuropilin-1 ligands. We propose that neuropilin-1 may act as a novel receptor on stromal cells by mediating interactions between stroma and primitive hematopoietic cells.     

Gene-expression and in vitro function of mesenchymal stromal cells are affected in juvenile myelomonocytic leukemia

An aberrant interaction between hematopoietic stem cells and mesenchymal stromal cells has been linked to disease and shown to contribute to the pathophysiology of hematologic malignancies in murine models. Juvenile myelomonocytic leukemia is an aggressive malignant disease affecting young infants. Here we investigated the impact of juvenile myelomonocytic leukemia on mesenchymal stromal cells. Mesenchymal stromal cells were expanded from bone marrow samples of patients at diagnosis (n=9) and after hematopoietic stem cell transplantation (n=7; from 5 patients) and from healthy children (n=10). Cells were characterized by phenotyping, differentiation, gene expression analysis (of controls and samples obtained at diagnosis) and in vitro functional studies assessing immunomodulation and hematopoietic support. Mesenchymal stromal cells from patients did not differ from controls in differentiation capacity nor did they differ in their capacity to support in vitro hematopoiesis. Deep-SAGE sequencing revealed differential mRNA expression in patient-derived samples, including genes encoding proteins involved in immunomodulation and cell-cell interaction. Selected gene expression normalized during remission after successful hematopoietic stem cell transplantation. Whereas natural killer cell activation and peripheral blood mononuclear cell proliferation were not differentially affected, the suppressive effect on monocyte to dendritic cell differentiation was increased by mesenchymal stromal cells obtained at diagnosis, but not at time of remission. This study shows that active juvenile myelomonocytic leukemia affects the immune response-related gene expression and function of mesenchymal stromal cells. In contrast, the differential gene expression of hematopoiesis-related genes could not be supported by functional data. Decreased immune surveillance might contribute to the therapy resistance and progression in juvenile myelomonocytic leukemia.     

Getting blood from bone: An emerging understanding of the role that osteoblasts play in regulating hematopoietic stem cells within their niche

Blood and bone are dynamic tissues that are continuously renewed throughout life. Early observations based upon the proximity of bone and hematopoietic progenitor populations in marrow suggested that interactions between skeletal and hematopoietic elements are likely to be crucial in the development and function of each system. As a result of these morphologic observations, several groups have demonstrated that the osteoblasts play an important role in hematopoiesis by serving as a specific local microenvironment, or niche, for hematopoietic stem cells. Significant new developments in this area of active investigation have emerged since our last examination of this area in 2005. Here we discuss these new insights into the function and morphology of the hematopoietic stem cell niche, with a particular focus on cells of the osteoblastic lineage.     

Constitutive expression of leukemia inhibitory factor RNA by human bone marrow stromal cells and modulation by IL-1, TNF-alpha, and TGF-beta.

Recent in vitro studies indicate that bone marrow mesenchymal elements, residing in close proximity to hematopoietic cell populations, elaborate a network of cytokines that are, at least partially, responsible for modulating the growth and maturation of the latter compartment. Leukemia inhibitory factor (LIF), a molecule with both positive and negative regulatory activities, has been implicated in murine embryogenesis and hematopoiesis. We demonstrate that cultured normal human bone marrow stromal cells constitutively express LIF message. Further, exposure of these cells to other hematopoietic modulators including interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) (but not interferon-alpha [IFN alpha]) increases the level of LIF RNA. Interestingly, cultured stromal cells derived from three of four patients with chronic myelogenous leukemia showed enhanced LIF expression. These observations suggest that LIF may participate, either alone or through interaction with other cytokines, in the bone marrow microenvironment-mediated influence on both normal and malignant hematopoietic processes.     

Natural estrogens enhance the engraftment of human hematopoietic stem and progenitor cells in immunodeficient mice

Hematopoietic stem and progenitor cells (HSPC) are crucial in the maintenance of lifelong production of all blood cells. These stem cells are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the recovery after HSPC transplantation. Transplantation efficacy can be limited by inadequate hematopoietic stem cell number, poor homing, low level of engraftment, or limited self-renewal. As recent evidence indicates that estrogens are involved in regulating hematopoiesis, we sought to examine whether natural estrogens (estrone or E1, estradiol or E2, estriol or E3 and estetrol or E4) modulate human HSPC. Our results show that human HSPC subsets express estrogen receptors, and that signaling is activated by E2 and E4 on these cells. Additionally, these natural estrogens cause different effects on human progenitors in vitro. We found that both E2 and E4 expand human HSPC. However, E4 was the best tolerated estrogen and promoted cell cycling of human hematopoietic progenitors. Furthermore, we found that E2 and, more significantly, E4 doubled human hematopoietic engraftment in immunodeficient mice without altering other HSPC properties. Finally, the impact of E4 on promoting human hematopoietic engraftment in immunodeficient mice might be mediated through the regulation of mesenchymal stromal cells in the bone marrow niche. Collectively, our data demonstrate that E4 is well tolerated and enhances human reconstitution in immunodeficient mice directly, by modulating human hematopoietic progenitor properties, and indirectly, by interacting with the bone marrow niche. This might have particular relevance for improving hematopoietic recovery after myeloablative conditioning, especially when limited numbers of HSPC are available.     

Minireview: The stem cell next door: skeletal and hematopoietic stem cell "niches" in bone

Long known to be home to hematopoietic stem cells (HSC), the bone/bone marrow organ and its cellular components are directly implicated in regulating hematopoiesis and HSC function. Over the past few years, advances on the identity of HSC "niche" cells have brought into focus the role of cells of osteogenic lineage and of marrow microvessels. At the same time, the identity of self-renewing multipotent skeletal progenitors (skeletal stem cells, also known as mesenchymal stem cells) has also been more precisely defined, along with the recognition of their own microvascular niche. The two sets of evidence converge in delineating a picture in which two kinds of stem cells share an identical microanatomical location in the bone/bone marrow organ. This opens a new view on the manner in which the skeleton and hematopoiesis can cross-regulate via interacting stem cells but also a novel view of our general concept of stem cell niches.     

Chemotherapy-induced mesenchymal stem cell damage in patients with hematological malignancy

Hematopoietic recovery after high-dose chemotherapy (HDC) in the treatment of hematological diseases may be slow and/or incomplete. This is generally attributed to progressive hematopoietic stem cell failure, although defective hematopoiesis may be in part due to poor stromal function. Chemotherapy is known to damage mature bone marrow stromal cells in vitro, but the extent to which marrow mesenchymal stem cells (MSCs) are damaged by HDC in vivo is largely unknown. To address this question, the phenotype and functional properties of marrow MSCs derived from untreated and chemotherapeutically treated patients with hematological malignancy were compared. This study demonstrates a significant reduction in MSC expansion and MSC CD44 expression by MSCs derived from patients receiving HDC regimens, thus implicating potential disadvantages in the use of autologous MSCs in chemotherapeutically pretreated patients for future therapeutic strategies. The clinical importance of these HDC-induced defects we have observed could be determined through prospective randomized trials of the effects of MSC cotransplantation on hematopoietic recovery in the setting of HDC with and without hematopoietic stem cell rescue.