Mesenchymal stem cell: use and perspectives - The Hematology Journal

The Hematology Journal (2003) 4, 92–96

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REVIEW

Mesenchymal stem cell: use and perspectives Angelo Tocci*,1 and Laura Forte1 1

Laboratory for Stem Cell Studies, Bambino Gesu` Research Hospital, Rome, Italy

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. The Hematology Journal (2003) 4, 92–96. doi:10.1038/sj.thj.6200232 Keywords:

microenvironment; mesenchymal stem cells; therapy

Definition of bone marrow (BM) stroma and mesenchymal stem cells (MSC) BM stroma function gives hematopoietic stem cells (HSC) the microenvironment appropriate for selfrenewal and/or prompt differentiation into hematopoietic progenitor cells (HPC) then into terminal specialized cells. In 1977, Dexter et al.1 set up culture conditions that could maintain and differentiate murine HSC for several weeks (long-term cultures, LTC). The start of Dexter-like cultures is dependent upon the establishment of a heterogeneous adherent layer. The Dextertype adherent layer was regarded initially as ‘physiological’, similar to ‘true’ BM microenvironment, and capable of sustaining the self-renewal and differentiation of repopulating HSC. Later studies demonstrated that: (i) the composition of Dexter-type stroma differs from that of physiological stroma; as an example, Dexter-type stromal layer is composed mainly of fibroblasts, macrophages, adipocytes, endothelial cells and smooth muscle cells, while the physiological stroma lacks smooth muscle cells2; (ii) unlike BM stroma in vivo, cells sustaining hematopoiesis in Dexter-type cultures are generally regarded as non-repopulating HSC, *Correspondence: A Tocci, Laboratory for Stem Cell Studies, Bambino Gesu` Research Hospital, Largo S. Onofrio 4, 00161 Rome, Italy; Tel: þ 390639725423; Fax: þ 390639725423; E-mail: [email protected] Received 22 April 2002; accepted 23 January 2003

although the exact relationship between long-term culture-initiating cells (LTC-IC) and repopulating HSC is currently unclear3 and (iii) although appropriate adaptations of the culture conditions make it possible to maintain human HSC for some weeks, the efficiency of the human systems proved to be much lower than that identified for murine HSC. This has prompted several studies on the composition of human BM. Human BM stroma has been dissected into two main components, the hematopoietic and the non-hematopoietic ones. In humans, the former includes HPCderived cells, mainly macrophages, while the latter, still poorly characterized, is composed mainly of endothelial and mesenchymal stem cells (MSC) with their derivatives (adipocytes, chondrocytes, cells of the osteogenic lineage). Classic studies have demonstrated the presence of fibroblast-like cells, termed colony forming unitfibroblast (CFU-F) in human BM4. The CFU-F is a clonogenic precursor, capable of limited self-renewing, that can differentiate into various connective tissue lineages. Recently, renewed interest on human BM stroma has increased and cells similar to the CFU-F have been isolated from adult human BM harvested from iliac crest and termed MSC. Recently, these cells have been better characterized as homogeneous cells; unlike CFU-F, extensive self-renewal and differentiation potential have been claimed to characterize MSC.

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Moreover, a panel of monoclonal antibodies (MoAbs) raised against some epitopes expressed on their surface allows phenotypic characterization of these cells.5

Methods of MSC isolation, morphological and phenotypic characterization Human MSC are obtained from BM harvested from adult iliac crest. Frequency of MSC in adult BM is reported to be 1 105–1 106 nucleated cells, as evaluated by CFU-F assay.4 In peripheral blood (PB), poorly characterized adherent cells, sharing some characteristics of MSC, have been reported in adult blood in G-CSF-treated patients and healthy donors;6,7 however, the presence of MSC in PB has not always been confirmed.8 MSC presence has been recently reported in term cord blood (CB), where 30% of cells express markers usually associated with MSC in no more than 30% of samples, with a higher frequency in preterm CB.9 Others have found poorly characterized stromal cells in term CB, but as for PB, the data have not always been confirmed.10 Interestingly, circulating MSC have been reported recently in human embryonic blood, fetal liver and BM at a frequency of approximately 10 106 nucleated cells in each district.11 Technically, it is possible to obtain 50–375 million cells from a 10 ml BM aspirate from adult donors.5 To isolate MSC from a BM aspirate, CB or PB, the samples are fractionated by density gradient for mononuclear cell isolation, resuspended in appropriate culture medium containing selected batches of fetal bovine serum (FBS) and allowed to adhere to plastic dishes for 2 days; then, nonadherent cells are removed and the remaining cells allowed to grow for 2–3 weeks. Cells initially generate a heterogeneous adherent cell layer including fibroblast-like and small round-shaped cells, while they appear uniformly spindle shaped after several passages in culture. Confluent cells are trypsinized and allowed to expand for as many as 40 generations without loss of multipotentiality. The MSC-derived cells are positively stained by May–Gru¨nwald Giemsa, alkaline phospha-

tase and adipocytic staining and appear as fibroblastlike spindle-shaped cells. A panoply of surface antigens has been reported to be expressed on MSC although there is no agreement on phenotypical characterization in the published literature (Table 1). Furthermore, most data derive from cells expanded in culture, which may have acquired a certain degree of commitment towards a differentiation pathway, compared to fresh cells. However, a simple panel of MoAbs can be used to characterize MSC by FACS analysis: they stain positively with anti-SH2 (CD105, endoglin), SH3 (CD166, ALCAM), SH4 (CD73) and STRO-1, while they do not express the CD45 (hematopoietic cells), the CD34 (hematopoietic progenitors, endothelial cells) and the CD14 (monocytes–macrophages).5 Since MSC phenotype is not univocal, morphology and retrospective functional assays are required to confirm the identification of MSC in order to use them for further functional evaluation. Interestingly, most MSC can be grossly distinguished from hematopoietic cells at FACS analysis, with a different distribution of size and granularity, being larger and more granular compared to common lymphocytes or CD34 þ cells.

Functional and differentiation properties of MSC Functionally, adult MSC are characterized by a doubling time of 33 h. They have a large expansive potential, and cell cycle studies reveal a subset (20%) of quiescent cells.12 The existence of a subset of quiescent cells in MSC cultures seems to be extremely significant, since their number and properties should be sufficient to sustain a steady supply of cells, which upon proliferation and commitment may serve as precursors for a number of nonhematopoietic tissues. MSC constitutively secrete several cytokines reviewed in Azizi et al.13 Many cytokines relevant in HSC proliferation and differentiation have been found among them (interleukin-6, Flt-3 ligand, stem cell factor, G-CSF, GM-CSF). Interestingly, MSC express several adhesion-related antigens (CD166, CD54,

Table 1 Immunophenotype of MSC as reported in the published literature Antigens CD49d, CD1a,CD11a, CD62E, CD62P, CD31, CD25, CD80, CD86, CD34, CD14, CD45, CD62 L, HLA-DR, CD71, CD58, CD102, CD50, CD106, CD127, HLA-ABC, CD123, CD120a, CD120b, CD117 CD49b,CD49 e, CD105 ( SH2), CD166 (SH3), CD44, CD 54 (ICAM-1), CD13, CD14, CD31, CD90, CD73 (SH3), CD73 (SH4), CD29, CD45, CD62L, HLA-DR, CD71, CD58, CD102, CD50, CD106 (VCAM-1), CD127, HLAABC, CD123, CD120a, CD120b, ASMA, MAB 1470

Source

Expression

References

Adult marrow, umbilical cord blood, first-trimester fetal blood, liver, bone marrow and pediatric bone marrow

Negative

5, 6, 20, 17, 16, 11, 15, 21, 9

Adult marrow, umbilical cord blood first-trimester fetal blood, liver and pediatric bone marrow

Positive

5, 6, 7,15, 16, 20, 17, 11, 9, 18

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CD31, CD106) and integrins (CD49, CD29, CD11, b4integrin),5 known to be relevant in sustaining the adhesion and proliferation of HSC. MSC support human LTC-IC, so they can provide a feeder layer for cultured HSC.14 BM MSC can sustain in vitro long-term hematopoiesis and, in combination with exogenously added cytokines (SCF, Flt-3 ligand and thrombopoietin), are able to support the expansion of clonogenic hematopoietic cells.15 MSC can replicate as undifferentiated cells and have the potential to differentiate into several different lineages of mesodermal origin, such as cartilage, bone, fat, tendon, muscle, myocardium and marrow stroma, both in vivo and in vitro, upon culture with appropriate combination of growth factors and chemicals.5,19,20 Recently, pluripotent cells have been isolated from adult BM and demonstrated to be able, at single cell level, to differentiate widely into both mesenchymal cells and cells with visceral mesoderm, neuroectoderm and endoderm properties. These cells termed multipotent adult progenitor cells are able to proliferate extensively without senescence, being an ideal candidate for clinical applications.21 MSC can differentiate into the osteogenic lineage by treatment with dexamethasone, 10% FBS, b-glycerol phosphate and ascorbate. After 1 week, a calcium accumulation is observed and alkaline phosphatase activity increases 4–10-fold.22 Interestingly, the bone morphogenic protein (BMP)-2, a peptide originally identified in ectopic extraskeletal sites and capable of inducing bone and cartilage formation, can mediate the rapid differentiation of MSC into osteogenic cells inducing the expression of specific transcription factors. Adipogenic differentiation can be induced with 1methyl-3-isobutylxanthine, dexamethasone, insulin, and indomethacin.5 Cells containing lipid vacuoles are observed following staining with oil red-O solution after 2–3 weeks. These adipocytes remain healthy in culture for at least 3 months. Chondrogenic differentiation is observed when cells are exposed to the influence of transforming growth factor-b3.5 MSCs are previously centrifugated to form a pelleted micromass that develops in a multilayered matrix-rich morphology histologically showing increased proteoglycan-rich extracellular matrix. These chondrocytes express type II collagen detected with MoAb C4F6. Cultures can be maintained for 3 months. Chondrocyte-like lacunae can be observed in histological section. Wakitani et al.23 demonstrated that MSC can also differentiate into myoblasts and myotubes by treatment with 5-azacytidine and basic fibroblast growth factor. Fukuda19 demonstrated that MSC can also differentiate into cardiomyocytes and are useful in cardiovascular tissue engineering. Surprisingly, the MSC are also able to transdifferentiate into tissues of different embryonic dermal origin. Exposed to basic fibroblast factor, dimethylsulfoxide, b-mercaptoethanol, and butylated hydroxyanisole, MSC rapidly differentiate into cells expressing neuronal phenotype.24 These data have been confirmed in vivo by injecting MSC into lateral ventricules of mice, and finding glial protein expression into engrafted cells.25 The Hematology Journal

Therapeutic applications of MSC Preclinical studies in animals Promising results have been achieved in transplantation in animals, demonstrating that MSC-derived cells can promote allogeneic HSC engraftment.26 MSC have been characterized from feline BM.27 Chuah et al.28 have demonstrated the possibility to transduce canine and murine MSCs with factor VIII-retoviral vectors and achieve transient production of therapeutic levels of human factor VIII into SCID mice. Keating et al.29 demonstrated that MSCs can be transfected with a gene for the factor IX and that this protein can be secreted for 8 weeks after systemic infusion into SCID mice. In the sheep model, marked MSC can engraft into the recipient hematopoietic microenvironment upon injection and remain functional.30,31 In the subhuman primates model, MSC display immunoregulatory features by altering lymphocyte reactivity to allogeneic target cells.32 Overall, animal studies suggest that MSC cotransplantation may facilitate the level of HSC engraftment.

Clinical studies in humans Clinical applications of human MSC are evolving rapidly with the aim to improve hematopoietic engraftment, expanding HSC, preventing graft-versus-host disease (GVHD),33,34 correcting inborn metabolic errors and delivering a variety of therapeutic genes into the cells.35 MSC have been used to regenerate marrow microenvironment after myeloablative therapy.36 Koc et al.36 reported on the feasibility of allogeneic infusion of MSC after expansion. Initial clinical studies have been performed in pediatric patients with osteogenesis imperfecta, a genetic disorder caused by a collagene defect and characterized by painful fractures, retarded bone growth, osteopenia, deformities. Horwitz et al.37,38 have shown that, in allogeneic BM transplantation, donor MSC may engraft, resulting in increased BM density; these results, however, have been questioned as the authors have not been able to demonstrate more than 2% osteoblasts of donor origin, currently not considered entirely appropriate to repopulate the defective compartment. In orthopedic surgery, the effects of tridimensional matrices and scaffold for chondrogenic induction of cultured BM MSC have been studied in vivo in the presence of cytokines and recombinant human bone morphogenetic protein-2 (rhBMP-2).39,40 Allay et al.41 demonstrated the expression of the lacZ and IL-3 in vivo after retroviral transduction of BM-derived human osteogenic MSC. Retrovirally transduced MSC should be able to express constitutively lacZ gene and differentiate into osteoblast in ceramic cubes implanted into SCID mice. MSCs form bone in those cubes and stain blue with X-gal indicating b-galactosidase expression.


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The capacity of MSC to differentiate into cardiomyocytes and the recent suggestion of functional improvement of cardiac function in infarcted baboons and in humans indicate that this approach may be useful in reducing the complications of cardiac disease, although the demonstration of the efficacy of this approach is pending.42,43

Finally, studies on the clinical applications of MSC transplantation are still limited and need to be confirmed by stringent protocols; however, the proliferative and differentiative properties of MSC and their easy manipulation may disclose unexpected broad application of this technology.

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