Experimental Hematology
Volume 28, Issue 8 , Pages 875-884, August 2000

Mesenchymal stem cells:

Biology and potential clinical uses

Osiris Therapeutics, Baltimore, MD, USA

Received 3 May 1999; received in revised form 26 March 2000; accepted 28 March 2000.

Article Outline

Abstract 

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.

Keywords:  Stroma, Transplantation, Hematopoiesis, Mesenchymal stem cell

 

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Stromal cell system 

There has been an increasing interest in recent years in the stromal cell system, which includes the marrow-derived stromal cell that supports hematopoiesis, as well as the mesenchymal stem cell and its progeny, connective tissue cells such as osteocytes, chondrocytes, tenocytes, adipocytes, and smooth muscle cells. The stromal cell system, first described by Maureen Owen [1] in 1985, has been the subject of investigation in the fields of connective tissue engineering, cell transplantation, hematopoietic stem cell transplantation, and gene therapy.

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What constitutes stroma? 

There are three main cellular systems in the bone marrow: hematopoietic, endothelial, and stromal (with stromal cells loosely referring to the nonhematopoietic cells of mesenchymal origin). The stromal cell system as proposed by Owen was based on an analogy with the hematopoietic system, in which mesenchymal stem cells reside within the marrow, maintain a level of self-renewal, and give rise to cells that can differentiate into various connective tissue lineages, including the osteogenic lineage as described by Friedenstein [2] in 1980 as well as stromal tissues [3]. Four main cell types comprising the postnatal marrow stromal tissue are known to support hematopoiesis: macrophages, adipocytes, osteogenic cells, and “reticular cells.” This is in contrast to the in vitro adherent layer derived from long-term in vitro bone marrow culture [4] and consisting of fibroblastic cells, macrophages, adipocytes, endothelial cells, and smooth muscle cells. The latter are not present in the extravascular space of the bone marrow and appear only in arteriolar walls.

Within the in vivo stromal environment, alkaline phosphatase positive (ALP+) reticular cells associate closely with hematopoietic cells [5]. These ALP+ reticular cells are thought to originate from cells that were destined to differentiate into osteoblasts but are capable of forming stroma. The presence of adipocytes in the postnatal stroma is dependent on a number of factors: 1) stage of skeletal development, because adipogenesis progresses from the diaphyses to the epiphyses; 2) age, because the number of adipocytes increases with age; and 3) the level of hematopoiesis, because adipogenesis appears to correlate indirectly with hematopoietic cell mass, which usually is reflected in the number of ALP+ reticular cells present in the marrow. It has been suggested that ALP+ reticular cells and adipocytes are alternative phenotypes that are modulated by the marrow environment [6]. Recently, the multiple lineages (osteogenic, chondrogenic, and adipogenic) were shown to be clonally derived from a single mesenchymal stem cell [7], supporting the concept of the stromal cell; however, further study of in vivo plasticity of the lineages is required.

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Differentiation potential 

The differentiation potential of the stromal cell system has been widely studied with respect to the mesenchymal connective tissues, in particular bone [8] and cartilage. Human stromal cells that had been depleted of circulating hematopoietic cells by negative immunoselection with antibodies against monocytes/macrophages (anti-CD14), endothelial cells (anti-CD31), and lymphocytes (anti-CD11a/LFA-1) were shown to coexpress genes characteristic of the osteoblastic lineage (alkaline phosphatase, osteocalcin, and osteopontin) and adipocytic lineage (lipoprotein lipase), indicating that stromal cells were uncommitted precursor cells [9]. Human marrow stromal fibroblasts are capable of forming colonies in vitro in the presence of serum and at least four growth factors: platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), and epidermal growth factor (EGF) 10, 11. Pittenger et al. [7] reported that approximately one-third of the initial adherent bone marrow-derived stromal colonies are pluripotent and capable of differentiation into the osteogenic, chondrogenic, and adipogenic lineages as demonstrated by lineage specific in vitro assays (Fig. 1).

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  • Figure 1. 

    Culture-expanded human mesenchymal stem cells exhibit a spindle-shaped fibroblastic morphology following culture expansion ex vivo (top panel). Under appropriate inducing conditions, the culture will demonstrate adipogenic differentiation evidenced by fat globules, chondrogenic differentiation as measured by staining for type II collagen, or osteogenesis as seen by calcium conditions. Assays are described in Pittenger et al. [7]

Kuznetsov et al. [12] demonstrated that in vivo transplants of all multicolony-derived marrow stromal fibroblasts derived from multiple in vitro stromal cell colonies resulted in bone formation, whereas only 58.8% of fibroblasts formed bone. Kuznetsov and colleagues [13] recently presented evidence for a circulating osteogenic precursor in humans, although the circulating precursor demonstrates greater variability in clonogenic potential than precursors derived from bone and bone marrow. The reported precursor frequency of osteoblastic precursors in normal human bone marrow is approximately four per 100,000 nucleated cells (based on ALP+ staining) and appears to correlate negatively with age and the existence of disease states such as osteoarthritis [14]. Whereas bone marrow-derived precursors are able to give rise to osteogenic progeny as well as hematopoietic supportive stroma and adipocytes, marrow fibroblasts derived from bone tissue have been shown to exhibit only osteogenic potential [15]. Human mesenchymal stem cells maintain the potential to differentiate into the osteogenic lineage for up to approximately 40 doublings in culture, even after cryopreservation 16, 17. In vivo bone formation by marrow stromal fibroblasts has been determined to be dependent on both the in vitro culture conditions and the matrix used to implant the marrow stromal fibroblasts, with hydroxyapatite/tricalcium phosphate (HA/TCP) ceramics resulting in the most consistent bone formation [18].

The bFGF and bone morphogenetic protein 2 (BMP-2) have been shown to synergistically enhance in vivo bone formation of mesenchymal stem cells that were preexposed to the two factors and subsequently implanted on porous ceramic cubes compared to those mesenchymal stem cells that were exposed to either bFGF or BMP-2 alone [12]. BMPs have been postulated to play a role in the selective differentiation of mesenchymal precursors into either the osteoblastic or adipogenic lineage [19]. Selective blocking of the BMP receptor type 1B (BMPR-1B) resulted in differentiation into the adipocytic lineage rather than ostoeblastic differentiation, suggesting that expression of BMPR-1B is required for mesenchymal stem cell commitment to the osteoblastic lineage. Conversely, overexpression of BMBR-1A blocked adipogenic differentiation and promoted osteoblastic differentiation, suggesting that the temporal expression or loss of the BMP receptors may play a key role in determining the lineage commitment of the mesenchymal precursors into osteoblasts or adipocytes [20].

In attempts to utilize the stromal cell system to direct osteogenesis for the purposes of clinical tissue regeneration, marrow stromal fibroblasts from mice implanted on gelatin sponges were demonstrated to be capable of repairing a craniofacial defect [21]. Rat mesenchymal stem cells that were isolated from the long bones, culture expanded, and implanted on HA/TCP regenerated a critical size defect in the tibia of syngeneic rats [22]. Similar bone regeneration, determined by x-ray and histologic evaluation, was demonstrated by canine mesenchymal stem cells that were adhered to HA/TCP and implanted in critical size defects in the autologous femur 22, 23. Human mesenchymal stem cells that had been isolated from bone marrow, ex vivo culture expanded, loaded onto HA/TCP, and implanted into a critical tibial defect of immunocomprised rats regenerated normal bone on histologic evaluation [24]. The formation of cartilage by stromal cell precursors has been the focus of several recent publicatons 25, 26, 27, 28, 29. In vitro differentiation assays have been developed that demonstrated the chondrogenic potential of these bone marrow-derived mesenchymal precursors. In vivo implantation of cells derived from mesenchymal stem cells demonstrated the potential for chondrogenic repair 30, 31. There also have been recent reports of differentiation of mesenchymal stem cells into tendon [32].

In 1998, Ferrari et al. [33] reported that bone marrow was a source of myogenic precursors capable of forming new muscle. Although not characterizing this myogeneic precursor within the marrow population, more recent findings from Jackson et al. [34] identified the murine muscle satellite cell, characterized by dye exclusion properties, to be capable of reconstituting the hematopoietic lineages in irradiated transplant recipients. These data implicate a CD34+ precursor to the muscle satellite cell, suggesting either a lineage derived from the hematopoietic stem cell or that the mesenchymal stem cell expresses the CD34 marker and is not distinguished from the hematopoietic stem cell by these criteria.

Studies involving direct injection of mesenchymal stem cells into the rodent brain reported migration of cells with the brain and differentiation into GFAP+ glial populations. This approach has used xenogeneic transplant of human cells into the rat brain, as well as homologous mouse/mouse tracking studies 35, 36. These findings raise further intriguing questions about the plasticity across cell lineage boundaries previously thought to be distinct and await more rigorous cell fate and serial transplant experiments to model adult connective tissue stem cell hierarchy.

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Isolation and culture conditions for human mesenchymal stromal cultures 

The concept that adult hematopoiesis occurs in a stromal microenvironment within the bone marrow was first proposed by Dexter et al., leading to the establishment of in vitro culture conditions for long-term bone marrow culture (LTBMC). These studies demonstrated that an adherent stromal-like culture could support maintenance of hematopoietic stem cells (HSC) 1, 4. Similar adherent cell cultures supporting early B lymphopoiesis have been described [37] demonstrating full recapitulation of B-cell ontogeny from purified CD34+ hematopoietic precursors [38]. This demonstration of the essential role of stromal support in hematopoiesis shaped the view that cell–cell interactions in the marrow microenvironment were necessary for normal hematopoietic function and differentiation.

Stromal cell cultures often are defined as the nonhematopoietic adherent cell population obtained by direct plating of bone marrow. Marrow stroma may have relatively simplex or complex cellular compositions depending on the growth media or plating substrate used. The majority of reported conditions used relatively undefined media compositions containing fetal bovine serum (FBS) or other animal sera, thus limiting the study of physiologic signals required for efficient attachment and culture expansion. However, ex vivo culture results in consistently reproducible stromal cell cultures and have been evaluated in human clinical studies for support of autologous hematopoietic engraftment [39].

We reported standard conditions for generation of marrow-derived mesenchymal stromal cultures 24, 40, and similar protocols are the subject of a recent review [41]. In brief, a bone marrow aspirate is collected and processed using density gradient centrifugation, from which light-density cells are taken and plated at a standard plating density in a DMEM media base containing FBS. After allowing 2 days for adherence to noncoated polystyrene, nonadherent cells are removed, and a feeding schedule established for a 14-day primary expansion period of adherent colonies. At this time, near-confluent cultures can be processed further by trypsinization and expansion through sequential passages to confluency. Cells may be expanded as many as 40 generations while still retaining their multipotent mesenchymal lineage capability, although growth rates are reduced. The expanded mesenchymal stem cells do exhibit a finite lifetime and do not display properties of immortalized cells.

Variation between laboratories in the use of specific growth factors or inducers in propagating these stromal cultures has been reported and likely results in selective enrichment of progenitor differentiation. For example, maintenance of stromal cultures in the presence of dexamethasone is known to enhance lineage progression along the osteogenic or adipogenic lineages, which may explain the relatively complex patterns of cell morphology and differentiation seen in Dexter [4] or Whitlock-Witte [38] cultures. Quito et al. [42] reported striking differences between stromal cultures supporting hematopoietic activity when k-FGF (fibroblast growth factor 4) was used to propagate the stromal culture. Delayed senescence and extended proliferation, in addition to improved hematopoietic support, were seen in cultures expanded with k-FGF. Majumdar et al. [40] directly compared an expanded mesenchymal stromal culture to Dexter culture with respect to hematopoietic support capability. Continued investigation into subpopulations of mesenchymally derived cells and their support of in vitro hematopoiesis may help define key regulatory stages of hematopoiesis in vivo.

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Colony-forming unit fibroblast activity and in vitro assays for mesenchymal stromal cultures 

It is important to develop improved and standardized tools for the isolation and characterization of stromal cell activity. It has been proposed that the stromal population of marrow is derived from a mesenchymal stem cell 1, 40, 43, giving rise to the various mesenchymal lineages. Isolation techniques for obtaining this mesenchymal stem cell have been directed primarily toward physical enrichment or toward exploiting plating properties, with relatively few descriptive phenotypic markers available for cell separation.

A standard in vitro assay for mesenchymal tissue potential is the colony-forming unit fibroblast (CFU-F) assay 44, 45, in which cells obtained from bone marrow are plated at low density either directly or following gradient separation, expanded as an adherent population, and quantified by scoring individual foci or colonies presumed to be derived from a single precursor. Using this assay, several groups estimated the number of mesenchymal stem cells in marrow to be approximately one in 104 to one in 105 marrow mononuclear cells. The CFU-F assay has not been routinely successful for the detection of mesenchymal stem cells in peripheral blood and cord blood. A number of investigators further investigated the homogeneity of colonies obtained using the CFU-F assay. In vitro mesenchymal lineage differentiation assays have been used to character progeny of individual colonies. Using in vitro assays to determine the differentiation profile of individual colonies, several groups demonstrated subpopulations within the mesenchymal stromal population [46], including cells with osteogeneic potential only or those also maintaining chondrogenic or adipogenic lineage potential. Thus, mesenchymal lineage commitment at the genetic level may be occurring in the absence of obvious morphologic differences and reflected following specific lineage induction. Cells derived from individual colonies also have been tested in vivo using implanted diffusion chambers in a rat model [47]. These results are similar to those from in vitro differentiation studies, namely that although full mesenchymal differentiation potential is demonstrated by some colonies, others show restricted lineage potential.

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Stromal cell precursor phenotype 

Unlike the hematopoietic stem cell and its progeny, the phenotype of the cells comprising the stromal cell system, including the mesenchymal stem cell and its progeny, has not been well described. Early reports identifying a precursor cell for the mesenchymal tissues comprising stroma suggested a common precursor between the hematopoietic and mesenchymal lineages [48]. This target cell population was characterized as CD34+, CD38−, HLA DR−. These findings were based on flow cytometric sorting of human fetal liver cells. These results were later modified by the finding that the hematopoietic and mesenchymal precursors could be fractionated within the CD34+ fetal liver population based on expression of the CD50 surface marker. Accordingly, CD50−, CD34+ cells gave rise to the mesenchymal lineages with CD50+, CD34+ cells giving rise to the hematopoietic lineages [49].

The CD34 marker is not expressed by ex vivo culture expanded mesenchymal stem cells [7]. It is possible that the CD34 antigen is expressed by the mesenchymal stem cell when directly isolated from marrow but lost on culture expansion, or that the fetal-derived stem cell precursor promotes CD34 expression, which is not abundant on the adult mesenchymal stem cell in marrow. Recent reports from Simmons et al. [50] demonstrate the use of the Stro-1 antibody for enrichment of mesenchymal precursors. This antibody was shown to bind to all of the cells associated with CFU-F activity of adult human bone marrow 51, 52, although the antigen to which this antibody is directed has not been reported. This population can be compared to the fetal liver stromal precursor cell population isolated by Huang and Terstappen [48], described earlier. However, in adult human bone marrow, Stro-1 CFU-F activity is associated with reduced expression of CD34. In addition, some CFU-F activity is found with cells expressing undetectable levels of CD34. This indicates that the CD34 antigen is not a clear-cut marker of adult human mesenchymal stem cell activity and leaves open the question of lineage relationships between the mesenchymal stem cell and the hematopoietic stem cell. It may be important to contrast the surface phenotype of stromal precursors directly isolated from bone marrow to stem cells expanded ex vivo, where attachment to culture vessel substrate and expanded by fetal calf serum or growth factors may result in loss or gain of surface receptors.

Conditions have been described for enrichment of CFU-F using Stro-1 antibody 50, 53, 54. This enrichment process is partial, in that 90% of the Stro-1+ cells are glycophorin A+ or CD19+ (late-stage erythroblast or B cells, respectively) and the CFU-F activity is confined to the glycophorin A− population. Further flow cytometric analysis indicates that the CFU-F precursor is positive for Thy-1 (CDw90), VCAM-1 (CD106), CD29/CD49 (integrin family), CD10, CD13, and receptors for PDGF, EGF, insulin-like growth factor 1 (IGF-1), and nerve growth factor (NGF) [55]. Cells isolated from CFU-F colonies have been reported to be negative for hematopoietic markers such as CD34 and CD45. The SH2 antibody, originally isolated by Haynesworth et al. [56], was shown to specifically react with CFU-F present in human bone marrow. Gordon et al. [57] reported the ability to isolate a stromal precursor from human marrow using a peptide mimicking the receptor-binding domain of TGF-β. Cells that bound to a collagen matrix embedded with the peptide were expanded and successfully tested for mesenchymal lineage induction. Robledo et al. [58] showed that stromal cells express type I and type II TGF-β receptors, from binding studies using soluble TGF-β, and demonstrated the presence of endoglin on human stromal lines.

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Human mesenchymal stem cells express surface markers important in hematopoietic cell interaction 

Mesenchymal stem cells express numerous receptors important for cell adhesion with hematopoietic cells. A recent review of adhesion receptors regulating hematopoiesis was reported by Verfaillie [59]. Hematopoietic progenitors express a large number of receptors responsible for adhesion to the extracellular matrix. These include the integrin families responsible for binding to fibronectin, laminin, or collagen, that is, expression of intracellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), platelet endothelial cell adhesion molecule (PECAM), L-selectin, and P-selectin. In addition, CD40, which has an affinity for hyaluronic acid and fibronectin, and sialomucins such as CD34, CD43, and CD164 also are known to be expressed on these cells. Antibody blocking and other experiments have implicated the β integrins as important in hematopoietic cell tracking and retention by stroma. Recent experiments involving CD164 homotypic binding between hematopoietic progenitors and stromal cells have implicated this receptor as an important factor involved in cell homing [60]. Turner et al. [61] performed in vitro cell adhesion studies to examine the binding of five human CD34+ hematopoietic cell lines to either extracellular matrix components or bone marrow stromal cultures. In these studies, adhesion to matrix or stromal cultures could be partially inhibited by divalent cation chelation or RGD peptide competition, implicating integrin-dependent adhesion. Additionally, binding was sensitive to digestion by chondroitinase ABC, suggesting CD44-dependent binding interactions. Other investigators used anti-CD44 antibodies to block human stromal cell support of hematopoiesis and concluded that normal CD34+ stem cells require CD44-mediated signaling [62]. Therefore, it is valuable to define the expression of adhesion markers on stromal cell cultures. Table 1 illustrates a current understanding of the phenotype of in vitro expanded mesenchymal stromal cells.

Table 1. Phenotypic characterization of human mesenchymal stromal cultures
Common nameCD locusDetection
Adhesion molecules*
ALCAMCD166Pos
ICAM-1CD54Pos
ICAM-2CD102Pos
ICAM-3CD50Pos
E-selectinCD62ENeg
L-selectinCD62LPos
P-selectinCD62PNeg
LFA-3CD58Pos
Cadherin 5CD144Neg
PECAM-1CD31Neg
NCAMCD56Pos
HCAMCD44Pos
VCAMCD106Pos
Hyaluronate receptorCD44Pos
Growth factors and cytokine receptors*
IL-1R (α and β)CD121a,bPos
IL-2RCD25Neg
IL-3RCD123Pos
IL-4RCD124Pos
IL-6RCD126Pos
IL-7RCD127Pos
Inteferon γ RCDw119Pos
TNF-α-1RCD120aPos
TNF-α-2RCD120bPos
FGFR Pos
PDGFRCD140aPos
Transferrin receptorCD71Pos
Integrins*
VLA-α1CD49aPos
VLA-α2CD49bPos
VLA-α3CD49cPos
VLA-α4CD49dNeg
VLA-α5CD49ePos
VLA-α6CD49fPos
VLA-β chainCD29Pos
β4 integrinCD104Pos
LFA-1 α chainCD11aNeg
LFA-1 β chainCD18Neg
Vitronectin R α chainCD51Neg
Vitronectin R β chainCD61Pos
CR4 α chainCD11cNeg
Mac1CD11bNeg
Additional markers*
T6CD1aNeg
CD3 complexCD3Neg
T4, T8CD4, CD8Neg
TetraspanCD9Pos
LPS receptorCD14Neg
LewisXCD15Neg
—CD34Neg
Leukocyte common antigenCD45Neg
5′ terminal nucleotidaseCD73Pos
B7-1CD80Neg
HB-15CD83Neg
B7-2CD86Neg
Thy-1CD90Pos
EndoglinCD105Pos
MUC18CD146Pos
BST-1CD157Pos

* Data are from Pittenger et al. [7] and Azizi et al. [36], or are previously unreported communication.

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Mobilization of circulating mesenchymal stromal cells 

Investigators have tried to detect circulating cells exhibiting the multilineage mesenchymal stem cell phenotype. Chesney et al. [63] recently described a human circulating fibroblast-like cell, which has been termed a fibrocyte. These cells are reported to be CD34+, CD45+, CD13+, and to be capable of synthesizing collagen. In addition, these cells express class II HLA molecules, the costimulatory molecules CD80 and CD86, and the adhesion molecules CD11a, CD54, and CD58. These cells also were demonstrated to induce T-cell responses consistent with a dendritic cell function. Previous studies from this group associate this circulating cell with cell recruitment into wound sites and tissue repair processes [64]. Additional evidence for circulating mesenchymal stem cell populations was provided by Ferrari et al. [33], who reported that the transfer of genetically marked (β galactosidase) bone marrow led to genetically marked muscle cell progenitors. These precursors respond to signals for muscle regeneration with proliferation and differentiation into mature myoblasts, indicating that the mesenchymal muscle progenitor is derived from a bone marrow-derived precursor or stem cell. Fernandez et al. [65] reported detection of stromal cells in peripheral blood of granulocyte colony-stimulating factor (G-CSF) mobilized breast cancer patients; however, others reported the inability to detect stromal cells from such patients [39]. These studies identified a population of adherent cells that could be cultured and were CD106 (VCAM), CD54 (ICAM), SH2+, SH3+ but also CD34−, CD14−, and CD45−. These studies suggest that mobilization of the mesenchymal stromal cell precursor might be achievable.

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Factors produced by mesenchymal stromal cultures 

We and others demonstrated the utility of stromal cell cultures for support of hematopoietic cell expansion ex vivo [40]. Consistent with this finding is the expression of many growth factor activities associated with hematopoietic support by expanded mesenchymal stromal cultures (Table 2). Rafi et al. [66] reported that long-term hematopoiesis on bone marrow-derived endothelial cell cultures is dependent on soluble factors including G-CSF, GM-CSF, M-CSF, kit ligand, IL-6, FLK-2, and LIF.

Table 2. Expression of growth factors by human mesenchymal stromal cultures*
Expressed in long term bone marrow culture without induction
IL-6
IL-7
IL-8
IL-11
IL-12
IL-14
IL-15
LIF
M-CSF
Flt-3 ligand
SCF
Induced by IL-1
IL-1
IL-1β
IL-6
IL-8
IL-11
IL-11
G-CSF
GM-CSF
LIF
Not expressed
IL-2
IL-3
IL-4
IL-10
IL-13

* Summary of data reported in Azizi et al. [36]. RT-PCR detection of indicated genes in four individual human MSC cultures, cultured under standard isolation conditions, long-term bone marrow culture conditions, or induced with 10 to 100 U/ml IL-1.

In addition to providing critical cell–cell contact and producing growth factors for hematopoiesis, mesenchymal stem cells also may attract infused hematopoietic stem cells to the marrow by inducing homing receptors [67]. Peled et al. [68] assayed the influence of stromal derived factor 1 (SDF-1) chemokine in recruiting CD34+ cells to the marrow in an NOD/SCID model of human hematopoiesis. In these experiments, it was postulated that homing to bone marrow would be enhanced through the signaling of SDF-1 and its receptor CXCR4. In fact, these data indicated that there was significant enhancement of CD34+ cell migration and engraftment in marrow, which was dependent on the expression of CXCR4. CD34+ cell migration could be blocked using anti-CXCR4 antibody, and SDF-1 stimulated expression of CXCR4 could convert a poorly engrafting population of CD34+ cells into a cell population with enhanced engraftment potential. Kim and Broxmeyer [69] examined the chemotactic response to SDF-1 and steel factor and determined that both factors acted cooperatively in attracting CD34+ cells from cord blood and bone marrow. Thus, coinfusion of stromal cells with hematopoietic transplant in myeloablated cancer patients may accelerate recovery by stimulating CD34+ cell homing to marrow.

The stromal cell system also may play a role in the early maturation of T lymphocytes by providing necessary adhesion sites. Murine thymocytes plated onto a bone marrow stromal culture displayed differential sensitivity for adherence [70]. Greater than 60% of the double-negative T cells (CD4−/CD8−) were bound consistently during the incubation, with additional double-positive T-cell binding (22%). interestingly, neither single positive (CD4+ or CD8+) population showed significant binding. Continued feeding of the cultures resulted in steady production of replicating immature T cells, suggesting that bone marrow stroma may function as an extrathymic site of T maturation. Attempts have been made to induce T-cell differentiation using thymic stromal cultures with reported T-cell receptor gene rearrangement and the appearance of both early T and natural killer (NK) cell markers. Such cultures after longer periods of time resulted in preferential amplification of an immature NK cell pool [71]. It is likely that T-cell interaction with stroma or mesenchymal fibroblasts plays an important role in wound healing, as many of the T-cell cytokines modulate mesenchymal stromal cell function, including extracellular matrix production. Burger et al. [72] studied this effect by the coculture of synovial fibroblasts and dermal fibroblasts. They demonstrated that chronic exposure to stimulated T cells results in extracellular matrix catabolism. Plating whole human peripheral blood fractions onto mesenchymal stromal cultures in vitro showed less affinity for B cells and myeloid cells and higher affinity for activated T cells.

Genetically modified mesenchymal stem cells: potential for gene delivery 

The ability of mesenchymal stromal cells to self-renew at a high proliferative rate makes the stromal cell an excellent target for retroviral gene therapy. In a number of studies of gene transfer into stromal cell populations, it was demonstrated that adherent stromal cells can be transduced, with efficient and long-term expression [73]. In an early report, the genetically transduced stromal cells were implanted in an ectopic site in the mouse and underwent secondary transplantation with demonstration of 74% stable gene transfer efficiency [74].

The potential of stromal cells to serve as vehicles for gene delivery and protein production was reviewed by Clark and Keating [75] and by Gronthos and Simmons [54]. Dao and Nolta [76] reported the stable expression of IL-3–transduced human bone marrow stroma for at least 4 months in the xenogeneic immunodeficient (bnx) mouse model and supported human hematopoiesis from cotransplanted human CD34+ cells. A recent report confirmed the ability of murine bone marrow stromal cells transduced with the human IL-3 gene to secrete detectable human IL-3 in the NOD-SCID mouse model for up to 8 weeks [77]. The use of vector-modified stromal cells to secrete growth hormone or factor IX was examined in the nonablated, autologous canine model [78]. Because the authors used human genes in the immunocompetent canine system, it was not expected that the secreted proteins would be detected long term; however, vector-modified stromal cells were detected by polymerase chain reaction (PCR) for up to 15 weeks. The detection of factor IX protein persisted for 9 days after stromal cell infusion, suggesting that, in an optimized system, marrow stromal cells might demonstrate potential for adequate correction of some of these gene defects. A novel approach to the selection and subsequent retroviral transfection with the human factor IX gene of a population of murine marrow stromal cells binding a TGF-β1–von-Willebrand's factor fusion protein resulted in detectable levels of human factor IX in B6CBA mice [79]. High transduction efficiencies were reported for factor VIII gene transfection into human and murine marrow stromal cells, in particular using retroviral vectors containing the gibbon ape leukemia virus envelope [80].

Human mesenchymal progenitor cells were transduced with a marker gene (LacZ) and a gene for a secreted protein (IL-3) with an efficiency of 18% [81]. The transduced cells maintained the precursor phenotype, as evidenced by the ability to form bone in the in vivo osteogenic assay in SCID mice with evidence of LacZ expression in osteoblasts and osteocytes and detectable systemic human IL-3 expression. Another group confirmed the retroviral transduction of the bone marrow stromal cells with differentiation into bone and additionally demonstrated that local administration of adenoviral vectors in vivo to transfect the LacZ and neo(R) genes to a bony defect resulted in expression of the genes for up to 6 weeks [82]. These data would support a possible role for the use of mesenchymal stromal cells and gene therapy in the enhancement of fracture healing. Bone marrow cells that were genetically modified were transplanted into immunodeficient mice and were detected in muscle after having undergone myogenic differentiation [33], demonstrating the potential for gene therapy of the mesenchymal stem cell and its progeny.

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Transplantation of stromal cells using in vivo models 

Pereira and colleagues [35] and Prockop [83] demonstrated that, after adherence to plastic and ex vivo expansion in culture, mesenchymal cells from the bone marrow of transgenic mice expressing the human minigene for collagen I that were injected into irradiated isogenic mice could be detected in bone, cartilage, lung, spleen, and marrow 5 months later at levels approximating 1.5% to 12%. Reverse transcription PCR (RT-PCR) assays indicated that the human collagen I gene was expressed in a tissue-specific manner, thereby demonstrating that the ex vivo expanded mesenchymal cells can give rise to long-lasting connective tissue precursors. In a subsequent report [35], irradiated transgenic mice with an osteogenic imperfecta phenotype were the recipients of either wild-type bone marrow or mesenchymal stromal cells. DNA from the donor cells was detected consistently in bone, cartilage, and lung at 1 to 2 months. Azizi et al. [36] reported that human marrow-derived mesenchymal cells directly injected into rat brain engrafted without evidence of rejection or inflammation, suggesting that the brain also may be a target for in vivo use of stromal cells.

Cotransplantation of mesenchymal stem cells with HSCs has been reported to enhance HSC engraftment. Human ex vivo expanded marrow-derived mesenchymal stem cells were cotransplanted with human CD34+ isolated from cord blood into NOD-SCID mice and demonstrated a 10 to 20-fold increase in engraftment as determined by human CD45+ expression as compared to transplantation of the isolated CD34+ cells alone [84]. Dogs underwent autologous transplantation with canine G-CSF mobilized peripheral blood progenitor cells after single-dose lethal irradiation and cotransplantation with ex vivo expanded canine Mesenchymal stem cells that had been retrovirally transduced with the green fluorescent protein (GFP) transgene [85]. The dogs demonstrated good hematopoietic recovery after transplant and GFP+ cells were detected by DNA PCR and RT-PCR analysis at 6 months after transplant in bone marrow.

A potential role for stromal cell transplantation in allogeneic transplantation was addressed by a report in 1994 in which autoimmune mice (MRL/MP-Ipr/Ipr) underwent allogenic transplantation with cotransplantation of donor bone as a source of a stromal cell graft [86]. The mice survived for 48 weeks after transplantation without recurrence of any autoimmune phenomenon, even though the mice possessed radioresistant stem cells and typically experiences recurrence of autoimmune disease within 5 months of allogeneic transplantation. The authors suggest that the stromal cells not only prevent the occurrence of graft failure, but play a role in the complex immunoregulation of T and B cells. Recently, osteoblasts isolated from murine long bones were cotransplanted with extensively selected murine marrow stem cells into fully allogeneic mouse strains [87]. The mice were fully reconstituted with allogeneic donor cells and demonstrated good immunologic reconstitution, thus suggesting that bone progenitors or osteoblasts may play an essential role in graft facilitation in the allogeneic environment. Ex vivo expanded mesenchymal stem cells were found to exhibit a suppressive effect on T-lymphocyte responses in a mixed lymphocyte response in vitro [88]. Dogs underwent DLA matched allogeneic marrow transplantation after single-dose lethal irradiation and cotransplantation with ex vivo expanded canine mesenchymal stem cells that had been retrovirally transduced with GFP [89]. The dogs demonstrated full hematopoietic recovery, with a mean time to platelet recovery of 20 days. GFP+ cells were detected by DNA PCR analysis at 6 weeks after transplant in bone marrow, demonstrating the ability of allogeneic mesenchymal stromal cells to engraft recipient marrow.

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Role of stromal cells in the clinical setting 

Allogeneic bone marrow transplantation in osteogenesis imperfecta patients resulted in 1.5% to 2.0% engraftment of donor osteoblasts, suggesting that mesenchymal precursors present in the marrow may have a potential therapeutic role [90]. Intravenous infusions of ex vivo expanded human marrow-derived mesenchymal stem cells at a dose of up to 50 × 106 per patient were well tolerated by volunteers [91] and determined to be safe. A clinical trial in breast cancer patients undergoing autologous transplantation with peripheral blood stem cells and 1 × 106/kg mesenchymal stem cells was completed at Case Western University [92]. The MSC infusions were accompanied by no adverse events, and neutrophil (>500/μL at a mean of 8 days) and platelet (>50,000 μL at a mean of 13.5 days) engraftment was rapid with respect to historical controls. Early studies in the use of mesenchymal stem cells in autologous and in allogeneic transplantation are under way. These studies are paving the way for further applications of the stromal cell system in the field of transplantation with respect to hematopoietic support, immunoregulation, and graft facilitation.

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Acknowledgements 

We wish to thank Margaret Coddington for her excellent help with the manuscript, and we would like to thank our academic collaborators and the scientists at Osiris for their contributions and continued enthusiasm.

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References 

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PII: S0301-472X(00)00482-3

Experimental Hematology
Volume 28, Issue 8 , Pages 875-884, August 2000

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