Circulating mesenchymal stem cells

The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

Review

Circulating mesenchymal stem cells C. A. Roufosse a,∗ , N. C. Direkze b , W. R. Otto b , N. A. Wright b,c a

c

Department of Histopathology, Imperial College of Science, Medicine and Technology, Hammersmith Campus, DuCane Road, London W12 0NN, UK b Histopathology Unit, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Department of Histopathology, Bart’s and the London Hospital, Queen Mary’s School of Medicine and Dentistry, London E1 2AD, UK Received 5 August 2003; received in revised form 8 October 2003; accepted 14 October 2003

Abstract Mesenchymal precursor cells (MPCs) are multipotent cells capable of differentiating into various mesenchymal tissues, such as bone, cartilage, fat, tendon and muscle. They are present within both mesenchymal tissues and the bone marrow (BM). If marrow-derived MPCs are to have a role in repair and fibrosis of mesenchymal tissues, transit of these cells through the peripheral blood is to be expected. Although there is evidence for the existence of MPCs within the peripheral blood, results are debated and are not always reproducible. Variations in the methods of cell purification, culture and characterisation may explain the inconsistent results obtained in different studies. © 2003 Elsevier Ltd. All rights reserved. Keywords: Mesenchymal stem cell; Mesenchymal precursor cell; Circulating stem cell; Peripheral blood precursor cell; Peripheral blood stem cell

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What are mesenchymal stem cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Do mesenchymal progenitor cells circulate in the peripheral blood? . . . . . . . . . . . . . . . . . . . . . . . 3.1. Adult circulating mesenchymal stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Embryonal circulating mesenchymal stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Transplantation studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Solid organ transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Bone marrow transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Circulating BM-derived endothelial precursor cells (hemangioblasts) . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3.

586 586 588 589 591 592 592 593 593

Abbreviations: BM, bone marrow; BMPCs, blood mesenchymal progenitor cells; BMPRs, bone morphogenetic protein receptors; CB, cord blood at full term delivery; DMEM-LG, low-glucose Dulbecco’s MEM; GFP, green fluorescent protein; FCS, fetal calf serum; EC, endothelial cell; FISH, fluorescence in situ hybridization; GM-CSF, granulocyte–monocyte colony stimulating factor; HSC, haemopoietic stem cell; MAPCs, multipotent adult progenitor cells; MPCs, mesenchymal precursor cells; MSCs, mesenchymal stem cells; LDL, low density lipoprotein; PBFs, peripheral blood fibrocytes; PBPCs, peripheral blood precursor cells; PBSC, peripheral blood stem cell ∗ Corresponding author. Tel.: +44-20-8383-2445; fax: +44-20-7269-3491. E-mail address: [email protected] (C.A. Roufosse). 1357-2725/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2003.10.007

586

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

6. Do circulating MAPCs exist? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The study of adult type stem cells is a hot subject, because of the insights they offer into the understanding of tissue repair and regeneration, and also because they represent a possible alternative to embryonic stem (ES) cells in various therapeutic applications. Although haematopoietic stem cells are the best characterised, adult organisms contain several other types such as neural, epithelial and mesenchymal stem cells (MSCs). As will be discussed below, MSCs are found both in mesenchymal tissues and within the bone marrow (BM). Recent investigations have shown that after systemic infusion, marrow-derived MSCs engraft within multiple tissues of mesenchymal origin in the adult organism (Pereira et al., 1995; Prockop, 1997). How bone marrow-derived MSCs gain access to mesenchymal tissues, and whether they play a role in physiological turnover of these tissues, remains unknown. Transit of the MSCs through the peripheral blood would be expected to bridge the gap between the bone marrow and the mesenchymal tissues in need of repair. This article reviews current data on the existence of such a population of circulating MSCs.

2. What are mesenchymal stem cells? Excellent reviews of mesenchymal stem cells have already been published (Minguell, Erices, & Conget, 2001), but a brief summary will be provided here. “Mesenchyme” designates the developing loose connective tissue of an embryo, mainly derived from the mesoderm, and giving rise to a large part of the cells of the connective tissue in the adult. The definition is generally extended to include connective tissue cells in adult tissues such as (myo)fibroblasts, bone, cartilage, fat, tendon, muscles, and nerve tissue. Many mesenchymal tissues contain committed lineage-directed mesenchymal precursor cells (MPCs), which participate in local regeneration,

594 594 595 595

such as the satellite cell in skeletal muscle or the adipocyte progenitors of adipose tissue. Uncommitted mesenchymal progenitors, capable of giving rise to other types of mesenchymal tissues than the one they are present in, have also been found. Adult skeletal muscle contains, in addition to satellite cells, a more primitive population of cells of stellate morphology in culture, capable of differentiating along the lines of muscle, bone, cartilage and fat (Williams, Southerland, Souza, Calcutt, & Cartledge, 1999). Stromal-vascular cells of fat exhibit at least bipotent capacity, giving rise to both adipocytes and chondrocytes (Loncar, 1992). Clonal cell lines derived from bone cultures also exhibit multilineage differentiation (bone and fat) (Nuttall, Patton, Olivera, Nadeau, & Gowen, 1998). These data certainly provide evidence for the existence of uncommitted MPCs in several tissues, but the isolation of a true mesenchymal stem cell is more elusive. Mesenchymal stem cells represent a subset of precursor cells that adhere to the stem cell definition, i.e., they are capable of (1) self-renewal (ability to generate at least one daughter cell with characteristics similar to the to initiating cell); (2) multilineage differentiation from a single cell; and (3) in vivo functional reconstitution of the tissues to which they give rise (Verfaillie, 2002). The terms MPC and MSC are sometimes used interchangeably, and because most studies do not set out to prove strict adherence to the definition of “stem cell”, the term MPC will be used for most of this review. In addition to being present in mesenchymal tissues, MPCs have also been identified in the adult bone marrow. Marrow stromal cells were first studied for their role in supporting haemopoiesis, where they provide signals for differentiation and proliferation of haemopoietic stem cells and their progeny through direct cell–cell interactions and secretion of growth factors and chemokines (Cherry et al., 1994; Guerriero et al., 1997; Moreau et al., 1993). In culture as well, MPCs support growth of haematopoietic cells and infusion of MPCs along with haematopoietic stem cells

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

improves the efficiency of re-establishing functional haemopoiesis (Almeida-Porada, Flake, Glimp, & Zanjani, 1999; Almeida-Porada, Porada, Tran, & Zanjani, 2000; Anklesaria et al., 1987; del Canizo et al., 1999; Koc et al., 2000). Unexpectedly, cultures of adherent, spindle-shaped marrow-derived stromal cells were also found to exhibit multilineage mesenchymal differentiation, producing bone, cartilage and fat (Friedenstein, Gorskaja, & Kulagina, 1976). Numerous subsequent studies have refined and expanded these observations (Pittenger et al., 1999; Reyes et al., 2001). When mononuclear cells from bone marrow aspirates of human or rodent bone marrow are plated in a basal medium supplemented with fetal bovine serum, colonies of adherent fibroblast-like cells are observed. Depending on the specific culture conditions used (presence of growth factors, cytokines, etc.), these

587

cells are able to form bone, cartilage, tendon, muscle, fat, neural tissue and haemopoietic-supporting stroma (see Minguell et al., 2001). Variable results obtained from studies with different species, purification methods and culture techniques suggest that the marrow most probably contains a mixture of uncommitted and committed progenitor cells, capable of bi-lineage or tri-lineage differentiation. More difficult to demonstrate is the true marrow-derived mesenchymal “stem cell”, complying with the strict definition of a “stem cell” as defined above. Pittenger et al. (1999) rigorously analysed bone marrow MSCs in order to prove their adherence to the “stem cell” definition. They studied the progeny of colonies expanded from single adherent marrow cells, proving self-renewal and multilineage differentiation. This showed that at least some marrow cells represent true pluripotent stem cells rather than a mixture of committed progenitor

Fig. 1. Diagram of the proliferative hierarchy of mesenchymal progenitors. Adapted from Minguell et al. (2001). The figure is based on studies performed with expanded human bone marrow-derived mesenchymal progenitor cells. There are two main compartments: the uncommitted multipotent mesenchymal stem cells and the committed mesenchymal progenitor cells of decreasing stemness. Committed progenitors are named as colony forming units (CFU) and after their differentiation potential into S: haemopoietic-supporting stroma; O: osteoblasts; C: chondrocytes; T: tenocytes; A: adipocytes; skM: skeletal muscle; smM: smooth muscle; cM: cardiac muscle; As: astrocytes; Ol: oligodendrocytes; N: neurons. The third compartment represents the mature mesenchymal phenotypes.

588

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

Table 1 Main characteristics of bone marrow-derived MPCs Marker type

Designation

Specific antigens

SH2, SH3, SH4, STRO-1, ␣ smooth muscle actin, MAB1740

Cytokines and growth factors

Interleukins: 1␣, 6, 7, 8, 11, 12, 14, 15 LIF, SCF, Flt-3 ligand, GM-CSF, G-CSF, M-CSF

Cytokine and growth factor receptors

IL1-R, II-3R, II4-R, II-6R, II-7R, LIFR, SCFR, G-CSFR, IFN␥R, TNF1R, TNF2R, TGF␤1R, TGF␤2R, bFGFR, PDGFR, EGFR

Adhesion molecules

Integrins: ␣v␤3, ␣v␤5 Integrin chains: ␣1, ␣2, ␣3, ␣4, ␣5, ␣v, ␤1, ␤3, ␤4 ICAM-1, ICAM-2, VCAM-1, ALCAM-1, LFA-3, l-selectin, endoglin, CD44

Extracellular matrix

Collagen type I, III, IV, V and VI Fibronectin, laminin, hyaluronan, proteoglycans

Adapted from Minguell et al. (2001).

cells. Liechty et al. also demonstrated this capacity in an in vivo setting and showed that well-characterised human MSCs have the capacity to engraft into multiple organs in pre immune sheep (Liechty et al., 2000). MPCs isolated from the bone marrow by culture of adherent cells show considerable heterogeneity in terms of morphology and immunophenotype, and thus most probably represent a mixture of precursor cells derived from a small compartment of stem cells. Minguell et al. propose a scheme for the proliferative hierarchy for MPCs (see Fig. 1). Isolation and further characterisation of these various precursors is made difficult by the lack of well-defined specific antigenic markers for MPCs. Table 1 adapted from Minguell et al. shows the major antigenic determinants for MPCs. Stro-1 is an antibody made against marrow stromal cells (Simmons & Torok-Storb, 1991), which also stains pericytes. This antibody has been used to isolate an almost homogeneous population of non-cycling marrow MPCs, which have telomerase activity and the potential for multilineage differentiation (Gronthos et al., 2003). Other specific antigens for MPCs include SH2, SH3 and SH4 (Bruder, Jaiswal, & Haynesworth, 1997) as well as an antigen recognised by MAB 1470, also present on endothelial cells (EC) (Erices, Conget, & Minguell, 2000). SH2 has been further characterised and has been shown to react with endoglin (CD105), a member of the TGF-beta family, usually found on the endothelium of post-capillary venules (Barry, Boynton, Haynesworth, Murphy, & Zaia, 1999). Bone morphogenetic protein receptors I and II (BMPRs) are present on embry-

onal mesenchyme and postnatally on osteoblasts and chondrocytes. Antibodies to the BMPR IA and II have also been found to react with bone marrow stromal cells (Lecanda, Avioli, & Cheng, 1997). There is however no consensus on which of these markers is most useful for the identification and isolation of MPCs. A major question arising from the existence of MPCs in the marrow, is whether they constitute a source of multipotent cells capable of participating in the repair and regeneration of multiple tissues of mesenchymal origin in the adult organism. Investigations have shown that after systemic infusion, marrow-derived MPCs home to the bone marrow but are also found in multiple mesenchymal tissues (Pereira et al., 1995; Prockop, 1997). Engraftment of MPCs following systemic infusion does not prove that mobilisation of marrow-derived MPCs occurs naturally. The discovery of naturally circulating MPCs in the peripheral blood is in this respect of utmost importance.

3. Do mesenchymal progenitor cells circulate in the peripheral blood? Several studies address this issue by attempting to isolate MPCs from peripheral blood using culture conditions similar to those defined for bone marrowderived MPCs, in either adult or fetal organisms. These studies, which show conflicting results, will be described first.

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

Other studies have examined the fate of marrowderived cells after systemic infusion. The engraftment of these cells in various mesenchymal tissues provides indirect evidence for circulating MPCs. Similarly, so does the participation of recipient cells in repair and fibrosis of solid organs after transplantation. These studies will be discussed second. 3.1. Adult circulating mesenchymal stem cells Several studies have been carried out in humans and in animals. Initially, workers searched for MPCs in collections of peripheral blood precursor cells (PBPCs). PBPCs are haemopoietic presursor cells isolated from peripheral blood after mobilisation from the bone marrow by administration of granulocyte–monocyte colony stimulating factor (GM-CSF). The aim of the present studies was to investigate whether marrow stromal cells were also mobilised from bone marrow into blood in response to GM-CSF. This question is of potential clinical importance, following previous reports that infusion of marrow stromal cells along with haemopoietic precursors may enhance haemopoietic reconstitution (Lazarus, Haynesworth, Gerson, Rosenthal, & Caplan, 1995). Although the studies used very similar methods, different results were obtained: Fernandez et al. (1997) studied blood samples from 14 patients with breast cancer after chemotherapy and administration of G-CSF or GM-CSF, 5 ␮g/kg per day. Peripheral blood stem cells (PBSC) were isolated by apheresis from the blood for further study and treatment. Bone marrow from healthy donors for allogeneic transplantation was also studied. BM harvests and density separated mononuclear cells from apheresis were suspended in ␣-MEM containing 20% fetal calf serum (FCS) and

589

cultured in petri dishes. After 20 days, a near confluent population of adherent, fibroblast-like cells admixed with some large round cells was observed in 11/14 samples. The immunophenotype of the fibroblast-like cells and the round cells was identical. The cells were negative for haemopoietic and macrophage markers CD45 and CD14, did not express CD34 but did express MPC markers recognised by monoclonal antibodies SH2 and SH3, as well as fibronectin, collagen (I, III and IV), ICAM and VCAM. The content of circulating MSC in growth factor-mobilised PBSC harvests was measured by flow cytometry. The median percentage of CD14−/SH2+ cells in the apheresis products was 0.63%. As a control, mononuclear cells from the peripheral blood of three healthy donors did not generate any stromal cells in culture, whereas mononuclear cells from a non-breast cancer patient after 5 days of G-CSF stimulation contained a fraction of 0.11% SH2+ stromal cells. The authors concluded that stromal cells similar to those present in the bone marrow are present in human PBPC collections, but not in normal PB samples (Table 2). Lazarus, Haynesworth, Gerson, and Caplan (1997) were unable to reproduce these results in a similar study. Peripheral blood precursor cells were collected from patients suffering from cancer undergoing autologous (n = 11) infusion of PBPCs. PBPCs from healthy allogeneic donors (n = 3) were also studied. Patients and healthy donors had received 5–10 ␮g/kg per day s.c. G-CSF before collection of PBPCs. Some of the patients had already received chemotherapy as well. PBPCs were collected by leukapheresis, and suspended in low-glucose Dulbecco’s MEM (DMEM-LG) with 10% FBS. Normal bone marrow aspirates from the same three allogeneic donors were also analysed, using the same methods as controls.

Table 2 Publications on circulating adult MPCs Author and year of publication

Species

Samples studied

MSC detected in PB or PBPC

Fernandez et al. (1997) Lazarus et al. (1997) Wexler et al. (2003) Zvaifler et al. (2000) Kuznetsov et al. (2001) Huss et al. (2000) Wu et al. (2003)

Human Human Human Human Human, mouse, rabbit, guinea pig Dog Rat

PB, PBPC, BM PBPC, BM PBPC, BM Elutriated PB PB PB PB

Yes, No No Yes, Yes, Yes, Yes,

PB: peripheral blood; PBPC: peripheral blood precursor cells; BM: bone marrow.

PBPC

PB PB PB PB

590

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

Cultures of bone marrow aspirates, as expected, showed a population of adherent fibroblast-like cells with multilineage potential, exhibiting positivity for SH2, SH3 and SH4. These cells were also able to form bone in vivo when placed in porous calcium phosphate ceramic cubes in SCID mice. Cultures from PBPCs of cancer patients or normal donors yielded only a few adherent cells, which did not have the morphology of MPCs and did not form bone in vivo. Wexler et al. (2003) were also unable to demonstrate circulating MPCs. This group analysed human bone marrow, cord blood (CB, full term delivery) and peripheral blood stem cells. PBSCs were obtained from washings of the bags used for normal donor PBSC collections. Details of growth factor administration were not given. Cells from BM, CB and PBSCs were suspended in DMEM with 10% FCS in vented flasks. Within 10–21 days, a confluent layer of adherent monomorphic fibroblast-like cells was observed in the BM cultures. The cells could be passaged two times and reached confluence each time. In addition, their immunophenotype was that of MSCs (CD34, CD45 and CD14 negative; SH2 positive) and they exhibited multilineage differentiation (osteoblastic and adipogenic). Cultures from CB and PBSC showed a minimal non-confluent layer of adherent fibroblast-like cells that could not be passaged and exhibited a monocyte and macrophage immunophenotype (CD45 and CD14 positive). The different results obtained in the studies by Fernandez et al., on the one hand, and Lazarus et al. and Wexler et al., on the other hand, may be attributed to differences in the mobilisation procedure and cell preparation. Different growth factor regimens, leukapheresis procedures, and culture media may all contribute to the discrepancy. Fernandez et al. report that the presence of MPCs in the PBPC harvests is correlated to the amount of CD34+ cells, a factor that may also explain differences. Not all attempts to isolate MPCs from peripheral blood without prior growth factor mobilisation have been unsuccessful. Zvaifler et al. (2000) isolated adherent fibroblast-like cells from buffy coats of normal human blood (100 samples). The blood was submitted to an elutriation procedure, and eluate fractions were suspended in DMEM and 20% FCS. After 7–14 days of culture, adherent fibroblast-like cells admixed with

a smaller population of large round cells were found in eluate fractions 7 and 8. These “blood mesenchymal progenitor cells” (BMPCs) were once again negative for CD45, CD14 and CD34, and expressed SH2 (CD105, endoglin), vimentin, collagen I, BMP-R IA and BMP-RII. Stro-1 was expressed in the large cells only. In the appropriate culture conditions features of adipocytic and osteoblastic differentiation were noted. Kuznetsov et al. (2001) extended the observations using animal models. They isolated adherent and clonogenic cells of fibroblastic morphology from the peripheral circulation of four different adult mammal species (mouse, rabbit, guinea pig and human). Only two colonies of adherent fibroblastic cells were obtained from human venous blood or buffy coat concentrates. In the mouse, rabbit and guinea pig, these were more frequent. The culture media used was varied, but based on ␣-MEM. The human fibroblast colony cells were CD45, CD14 and CD34 negative, but did not express MPC markers SH2 or Stro-1. One of the two colonies isolated from humans exhibited in vivo bone formation after transplantation into subcutaneous pockets in immunocompromised mice. This was confirmed by using species-specific DNA-repetitive sequences as probes for in situ hybridisation. Adipocytic conversion of human blood-derived adherent cells could also be induced in both clones. This multipotent differentiation potential (osteo-adipo-fibrogenic) is similar to that of bone marrow-derived MPCs. Huss, Lange, Weissinger, and Kolb (2000) isolated CD34 negative adherent fibroblast-like cells from the peripheral blood of dogs. After these cells were immortalised with SV40 large T antigen and transfected with GFP, they were injected into irradiated dogs and were later found in the bone marrow as flattened cells lining the bone spicules. Taken together, these results support the existence of a small population of circulating MPCs, the isolation of which is clearly difficult and subject to variation depending on the methods of isolation and sorting of PB mononuclear cells or PBPCs, or on culture conditions. In addition, the MPC cells in the PB are defined mainly by their immunophenotype and morphology, while the absence of a well-defined immunophenotype for MPCs also makes study comparisons difficult. Two studies were able to show multilineage differentiation of the circulating MPCs (adipocytic and osteoblastic).

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

There is no evidence so far to support a functional role for these circulating cells in mesenchymal tissue repair. To help address this latter issue, animal models, such as that used in the study by Wu et al. (2003), are of interest. An aortic pouch graft filled with Matrigel was designed to capture circulating mesenchymal precursor cells in rats. This device allowed the isolation of a population of spindle cells from the circulating blood, which demonstrated multilineage differentiation in culture, generating cells of osteogenic, chondrogenic, adipogenic and myogenic types. These cultures do not appear to be based on single cells and therefore the presence of a mixture of progenitors could provide an alternative explanation for the multilineage differentiation seen. However, selected cells from the primary blood-derived mesenchymal progenitor colonies were then immortalised by transfection with an amphotropic murine leukaemia retroviral vector containing human papillomavirus genes and clonal cell lines derived. These clonal cells were then shown to exhibit multilineage differentiation. One of these clonal cell lines was transduced to express the lacZ marker gene. These cells were then injected intravenously into recipients of a cardiac allograft undergoing rejection. X-gal staining of cardiac tissue showed patches of beta-galactosidase positive cells within areas of fibrosis. Primary cultures of mesenchymal cells established from the heart allograft contained about 10% beta-galactosidase positive cells. The injected cells were also shown to home to bone marrow, where up to 24% of cultures of mesenchymal cells harvested from femurs were beta-galactosidase positive. This study provides compelling evidence for the existence of a pool of circulating mesenchymal precursor cells, capable of homing to the bone marrow and of colonising sites of organ damage.

591

3.2. Embryonal circulating mesenchymal stem cells During development, haemopoiesis is migratory, occupying several sites in the body of the developing fetus before confining itself to the bone marrow. This implies that both haemopoietic cells and their stromal support cells could transit through the peripheral blood (Table 3). However, as we have seen above, Wexler et al. (2003) were not able to identify circulating MPCs in cord blood from full term deliveries. In contrast, Campagnoli et al. (2001) looked for the presence of circulating MPCs in early fetal life (first trimester). Nucleated cells from fetal blood were diluted with 10% FBS with no additional growth factors, and plated. The differentiation abilities of single colony-derived adherent cells were studied by culturing cells under conditions used to determine the differentiation potential of MSCs in adults (Digirolamo et al., 1999). The circulating fetal MSCs were able to generate cells of osteogenic, chondrogenic and adipogenic lineages. In addition to this similarity of differentiation potential, the morphology and immunophenotype were similar to the adult counterparts. The immunophenotype of these cells was determined by flow cytometry and in situ immunocytochemistry, with the cells being CD45−, CD34−, CD14−; SH2+, SH3+, SH4+, and CD44+. Like adult MSCs, fetal MSCs are able to support long-term haemopoiesis in vitro. Fetal MSCs were also isolated from fetal bone marrow and liver and found to have the same features as those in the peripheral blood. This is consistent with the previous observation that mesenchymal cells co-localise with foci of haemopoiesis early in ontogeny (Tavian, Hallais, & Peault, 1999). SH3+ cells represented 0.33 ± 0.05% of circulating cells, 0.2% of which were colony-forming mesenchymal cells.

Table 3 Publications on circulating embryonal MPCs Author and year of publication

Species

Samples studied

MSC detected in CB

Wexler et al. (2003) Erices et al. (2000) Campagnoli et al. (2001)

Human Human Human

BM, CB, PBSC CB FPB, FCB, FBM, FL

No Yes Yes

CB: cord blood; PBSC: peripheral blood stem cells; FPB: fetal peripheral blood; FCB: fetal cord blood; FBM: fetal bone marrow; FL: fetal liver.

592

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

There were also MSCs in the blood samples from the second and third trimesters, albeit at significantly lower frequency. These results raise the possibility of using fetal MSCs for in utero therapy for genetic disorders. The transduction efficiency is high in circulating MSCs (Campagnoli et al., 2002). Erices et al. (2000) conducted similar experiments on cord blood samples from gestations of between 32 and 41 weeks. Mononuclear cells were suspended in ␣-MEM containing 20% FBS, then seeded in flasks. Two different types of adherent cells were observed: 76% of cultures were heterogeneous and contained large osteoclast-like cells, including multinucleated forms, whereas the remaining 24% were homogeneous and composed of cells of fibroblastic morphology. The latter exhibited osteogenic and adipogenic differentiation in the appropriate media. The immunophenotype was that of MPCs, with expression of SH2, SH3, SH4, MAB1470, ␣SMA, CD13, CD29, CD54, CD49c and absence of expression of myeloid or endothelial antigens. This group of cells thus displayed immunohistochemical and functional properties similar to bone marrow-derived MPCs. Most cultures of umbilical cord blood giving rise to these fibroblastic adherent cells were from earlier gestations (before 36 weeks). These studies support the existence of a population of circulating MPCs in the fetus, particularly in early gestation. As in adult organisms, the smaller amounts of circulating MPCs in later gestation may make it difficult to isolate them.

4. Transplantation studies As already mentioned above, in vivo experiments involving bone marrow transplantation and solid organ transplantation can provide indirect evidence for the existence of circulating MPCs. 4.1. Solid organ transplants Several studies in both humans and animal models have shown that the origin of a proportion of mesenchymal cells within a grafted solid organ can be traced to the recipient of the graft rather than the donor. This suggests colonisation of the engrafted organ by either mesenchymal cells or their precursors. The most likely mode of access of these cells to the

organ is the recipient’s blood perfusing the organ. For example, Quaini et al. (2002) and Laflamme, Myerson, Saffitz, and Murry (2002) demonstrated that a proportion of cardiac myocytes within transplanted human hearts were derived from the host using in situ hybridisation for the Y chromosome in sex-mismatched transplants. Fibroblasts and myofibroblasts are an important component of connective tissue all over the body, although normally sparsely distributed and inactive. Following tissue injury, (myo)fibroblasts can enter and proliferate within the injured area, and play a central role in wound repair and fibrosis. A portion of the (myo)fibroblasts involved in the scarring process are thought to be of local origin (proliferation of native fibroblasts or migration from adjacent tissues: see Yang et al., 2002). However, studies using fibrosis induced in solid organ transplants indicate that at least part of the myofibroblasts are of recipient origin, and therefore from outside of the engrafted organ. This has been shown in the case of interstitial fibrosis in the kidney (Grimm et al., 2001; Lagaaij et al., 2001; Poulsom et al., 2001), in scarring of the skin (Badiavas, Abedi, Butmarc, Falanga, & Quesenberry, 2003; Direkze et al., 2003) and in the infarcted fibrosed myocardium (Kuramochi et al., 2003). Similarly, fibroblasts and myofibroblasts in tumour stroma appear to be recruited from the circulation (Studeny et al., 2002). However the exact nature of the fibroblast precursor is unknown. Bucala, Spiegel, Chesney, Hogan, and Cerami (1994) identified within the peripheral blood a population of spindle-shaped cells termed peripheral blood fibrocytes (PBFs), which they believe to be a circulating precursor of tissue fibroblasts. These cells colonised wound chambers implanted in the subcutis of mice along with inflammatory cells. They have hybrid features, with haematopoietic cell surface markers and mesenchymal fibroblast-like properties, such as production of vimentin, collagens I and III, and fibronectin. Cultured PBFs also displayed a unique profile of cytokine, growth factor and chemokine production (Chesney, Metz, Stavitsky, Bacher, & Bucala, 1998), suggesting pro-inflammatory and pro-fibrotic properties. In summary, PBFs show the hallmarks of a circulating mesenchymal progenitor cell, capable of being recruited to sites of tissue damage by chemokines, and fulfilling the functions of a tissue fibroblast. Whether PBFs are the source of all

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

blood-derived fibroblast precursors is unknown. The immunophenotype of PBFs is clearly distinct from that of the circulating MPCs described in the adult and embryonal blood, and their relationship to these populations remains to be investigated.

593

recipients bone marrow, then participate in a normal biological cycle in which marrow MPCs serve as a source of progenitor cells for mesenchymal tissues.

4.2. Bone marrow transplants

5. Circulating BM-derived endothelial precursor cells (hemangioblasts)

Abundant studies address the issue of bone marrow stem cell plasticity, most of which are based on tracking the progeny of transplanted bone marrow cells. Transplanted whole bone marrow can differentiate into mesenchymal cell types such as skeletal muscle (Ferrari et al., 1998; Gussoni et al., 2002), cardiac muscle (Orlic et al., 2001), and others. Horwitz et al. (1999) reported that bone marrow transplantation in patients with osteogenesis imperfecta (deficiency collagen type I leading to fragile bones) resulted in clinical and radiological improvement. This was attributed to replacement of a small proportion (1.5–2%) of the patient’s defective osteoblasts by donor mesenchymal cells free from the genetic defect. Bone marrow transplantation studies also confirm the potential origin of (myo)fibroblasts from outside the site of injury. Sex-mismatched bone marrow transplantation has illustrated that whole bone marrow-derived cells can contribute to myofibroblast populations in the colon and small intestine in mice and humans (Brittan et al., 2002) as well as the lung (Direkze et al., 2003; Epperly, Guo, Gretton, & Greenberger, 2003), kidney, adrenal, hair follicle and stomach in mice (Direkze et al., 2003). This contribution is enhanced by injury and bone marrow-derived cells have also been shown to contribute to fibrosis (Direkze et al., 2003). A few studies investigate the progeny of systemically infused MPCs (Devine, Cobbs, Jennings, Bartholomew, & Hoffman, 2003; Pereira et al., 1995; Prockop, 1997). Cells derived from the MPCs were found in multiple tissues, such as bone and cartilage but also spleen, bone marrow and various solid organs such as lung and liver. The exact nature of the cells engrafted in these organs has not always been investigated. Toma, Pittenger, Cahill, Byrne, and Kessler (2002) were however able to demonstrate that systemically infused human MPCs give rise to cardiomyocytes in SCID mice. The results of these studies suggest that infused donor MPCs first populate the

If there is good evidence that bone marrow-derived cells can circulate and seed many tissues and organs, what of the possibility that these same, or similar, cells can also seed new or existing endothelium? The answer, broadly, is yes, and recent evidence is becoming stronger that this is a frequent event under certain circumstances. For example, it has been shown in vitro that cells derived from fetal bone marrow can differentiate into endothelial and haemopoietic cells (Guo et al., 2003). In adults CD34−, CD105+ mesenchymal stem cell lines can be derived from human peripheral blood that are potential haemopoietic progenitors given the appropriate environment (Conrad, Gottgens, Kinston, Ellwart, & Huss, 2002). The evidence for embryonic (Eichmann, Pardanaud, Yuan, & Moyon, 2002; Sainteny, 2001) and adult (Hirschi & Goodell, 2001) circulating haemangioblasts (precursors of both haemopoietic and endothelial cells) has been reviewed recently, so we confine ourselves to significant recent data. Such a report shows that haemopoietic stem cells (lin−, c-kit+, sca-1+) cells can transdifferentiate into functional endothelial cells in vivo. Transplanted adult BM HSC-derived GFP positive or sex-mismatched EC cells were found in several tissues (lung, heart, skeletal muscle, intestine, and in particular the liver portal vein), and importantly they were not the products of cell–cell fusion events, since they contained only 2N DNA, and were either GFP+ or male by FISH, having one Y and one X chromosome (Bailey et al., 2003). Cell fusion has been an issue of some controversy recently (Alison et al., 2003; Wells, 2002), so these observations are truly significant. The authors also showed EC functionality by infusing fluorescent LDL vesicles labelled with DiI, and showed uptake in 3 h into donor-derived GFP+ EC by double labeling. About 5% of EC were of donor origin at 6–8 months post-transplant. These experiments were repeated using a single GFP+ HSC cell infused together with Sca-1-negative BM cell support, with identical results,

594

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

indicating that true hemangioblast activity rests with the HSC fraction. In other recent reports (Cogle et al., 2003; Grant et al., 2002) a model of retinal neovascularisation in NOD/SCID mice infused with single human CD34+ cells from umbilical cord blood, showed human endothelial cells after retinal ischemia. Both these reports also conclude that true hemangioblast potential exists in the HSC fraction of the bone marrow. Another line of evidence suggesting that hemangioblast potential derives from the BM comes from data on the chronic myeloid leukemic (CML) patient. This condition is exemplified by the BCR-Abl fusion gene, which has been used as a marker of cell lineage in CML patients (Gunsilius, 2003). EC from these patients also contained the BCR-Abl fusion gene, which suggests that the diseased BM can also supply hemangioblast cells. The conclusions about hemangioblastic activity in the BM may have profound implications for tumor progression and metastasis in general. It is well established that tumors often induce blood vessel formation locally (Sullivan & Bicknell, 2003), but now that such inductions can arise from outside the tumour as well, this is truly a double whammy for the cancer patient. There is an urgent need for investigations into the ways hemangioblastic transdifferentiation can be either prevented, or otherwise subverted into a method for direct tumor attack from those BM-derived cells.

6. Do circulating MAPCs exist? A subset of marrow cells that co-purify with MPCs, termed multipotent adult progenitor cells (MAPCs) appear to have considerable plasticity, in that, in addition to generating mesenchymal cell types, they are also able to give rise to endothelial cells, neural cells and epithelial cells in vitro under defined culture conditions (Jiang et al., 2002a; Reyes et al., 2001). MAPCs have been purified from human, murine or rat bone marrow mononuclear cells. In vivo after injection into blastocysts, MAPCs contributed to most somatic tissues. In addition, infusion of MAPCs into peripheral blood by tail vein injection in mice is also followed by multilineage engraftment, in particular in organs with a high cellular turnover rate (bone mar-

row, spleen, liver, lung, intestine, lung, endothelium). No engraftment was found in skeletal or cardiac muscle. This was attributed to the low turnover of these organs under normal conditions. The hypothesis that MAPCs may represent an embryonic stem cell remnant has been put forward (Jiang et al., 2002b). In this concept, the bone marrow would contain a population of multipotent cells set-aside during embryogenesis for postnatal repair and remodelling of a variety of tissues. Cells with MAPC characteristics have also been found in the muscle and brain (Jiang et al., 2002b), prompting the hypothesis that MAPCs may circulate from the bone marrow to other tissues, transiting in the peripheral blood. Work is being done to investigate whether such circulating cells do exist. The existence of MAPC in vivo still needs to be proven: they could result from dedifferentiation of MPCs in vitro (Verfaillie, 2002).

7. Conclusions Mesenchymal stem cells have the potential to be a source of multipotent cells for autologous cell and gene therapy. Following isolation, purification and possibly in vitro differentiation, MPCs could be injected for local cell therapy in patients with damaged bone, cartilage or tendon (Minguell et al., 2001). Genetically modified MPCs could also be used to replace dysfunctional mesenchymal cells, as suggested in the study by Horwitz et al. mentioned earlier (Horwitz et al., 1999). Adult bone marrow is the most likely source of MPCs. Before such therapeutic applications become realistic, we must fill the gaps in our understanding of MPCs. We need to define more reproducibly the phenotype and morphology of MSCs, study the mechanisms controlling their mobilisation from bone marrow, their homing properties, as well as their differentiation programme to various tissue types and their physiological role. The studies summarised in this report provide support for the existence of a population of circulating MPCs in the peripheral blood, particularly following mobilisation of cells from the bone marrow by treatment with G(M)-CSF. The differing results obtained from one study to another highlight the need for a reproducible method of isolation of MPCs from the peripheral blood before any reliable conclusions can be reached. Once this is

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

accomplished, further investigations of MPCs in the blood following tissue injury or cytokine administration should provide insights into the mechanisms by which these cells may traffic to tissues in need of repair. Various attempts to increase mobilisation of the MPCs into the circulation may also prove useful for therapeutic applications.

Acknowledgements Dr. Candice Roufosse is supported by a Clinical Research Fellowship Grant from the Wellcome Trust. References Alison, M. R., Poulsom, R., Otto, W. R., Vig, P., Brittan, M., Direkze, N. C., Preston, S. L., & Wright, N. A. (2003). Plastic adult stem cells: Will they graduate from the school of hard knocks? Journal of Cell Science, 116(4), 599–603. Almeida-Porada, G., Flake, A. W., Glimp, H. A., & Zanjani, E. D. (1999). Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Experimental Hematology, 27(10), 1569–1575. Almeida-Porada, G., Porada, C. D., Tran, N., & Zanjani, E. D. (2000). Cotransplantation of human stromal cell progenitors into preimmune fetal sheep results in early appearance of human donor cells in circulation and boosts cell levels in bone marrow at later time points after transplantation. Blood, 95(11), 3620– 3627. Anklesaria, P., Kase, K., Glowacki, J., Holland, C. A., Sakakeeny, M. A., Wright, J. A., FitzGerald, T. J., Lee, C. Y., & Greenberger, J. S. (1987). Engraftment of a clonal bone marrow stromal cell line in vivo stimulates hematopoietic recovery from total body irradiation. Proceedings of the National Academic Science of United States of America, 84(21), 7681–7685. Badiavas, E. V., Abedi, M., Butmarc, J., Falanga, V., & Quesenberry, P. (2003). Participation of bone marrow derived cells in cutaneous wound healing. Journal of Cell Physiology, 196(2), 245–250. Bailey, A. S., Jiang, S., Afentoulis, M., Baumann, C. I., Schroeder, D. A., Olson, S. B., Wong, M. H., & Fleming, W. H. (2004). Transplanted adult hematopoietic stem cells differentiate into functional endothelial cells. Blood, 102, 13–19. Barry, F. P., Boynton, R. E., Haynesworth, S., Murphy, J. M., & Zaia, J. (1999). The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105). Biochemistry and Biophysics Research Community, 265(1), 134–139. Brittan, M., Hunt, T., Jeffery, R., Poulsom, R., Forbes, S. J., Hodivala-Dilke, K., Goldman, J., Alison, M. R., & Wright, N. A. (2002). Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon. Gut, 50(6), 752–757.

595

Bruder, S. P., Jaiswal, N., & Haynesworth, S. E. (1997). Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. Journal of Cell Biochemistry, 64(2), 278–294. Bucala, R., Spiegel, L. A., Chesney, J., Hogan, M., & Cerami, A. (1994). Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Molecular Medicine, 1(1), 71–81. Campagnoli, C., Roberts, I. A., Kumar, S., Bennett, P. R., Bellantuono, I., & Fisk, N. M. (2001). Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood, 98(8), 2396–2402. Campagnoli, C., Bellantuono, I., Kumar, S., Fairbairn, L. J., Roberts, I., & Fisk, N. M. (2002). High transduction efficiency of circulating first trimester fetal mesenchymal stem cells: Potential targets for in utero ex vivo gene therapy. BJOG, 109(8), 952–954. Cherry, Yasumizu, R., Toki, J., Asou, H., Nishino, T., Komatsu, Y., & Ikehara, S. (1994). Production of hematopoietic stem cell-chemotactic factor by bone marrow stromal cells. Blood 83(4), 964–971. Chesney, J., Metz, C., Stavitsky, A. B., Bacher, M., & Bucala, R. (1998). Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes. Journal of Immunology, 160(1), 419–425. Cogle, C. R., Wainman, D. A., Jorgensen, M. L., Guthrie, S. M., Mames, R. N., & Scott, E. W. (2004). Adult human hematopoietic cells provide functional hemangioblast activity. Blood, 103(1), 133–135. Conrad, C., Gottgens, B., Kinston, S., Ellwart, J., & Huss, R. (2002). GATA transcription in a small rhodamine 123(low)CD34(+) subpopulation of a peripheral blood-derived CD34(−)CD105(+) mesenchymal cell line. Experimental Hematology, 30(8), 887–895. del Canizo, M. C., Lopez, N., Vazquez, L., Fernandez, M. E., Ana, B., Caballero, M. D., Amigo, M. L., Mateos, M. V., Gutierrez, N., & San Migue, J. F. (1999). Hematopoietic damage prior to PBSCT and its influence on hematopoietic recovery. Haematologica, 84(6), 511–516. Devine, S. M., Cobbs, C., Jennings, M., Bartholomew, A., & Hoffman, R. (2003). Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates. Blood, 101(8), 2999–3001. Digirolamo, C. M., Stokes, D., Colter, D., Phinney, D. G., Class, R., & Prockop, D. J. (1999). Propagation and senescence of human marrow stromal cells in culture: A simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. British Journal of Haematology, 107(2), 275–281. Direkze, N. C., Forbes, S. J., Brittan, M., Hunt, T., Jeffery, R., Preston, S. L., Poulsom, R., Hodivala-Dilke, K., Alison, M., & Wright, N. A. (2003). Multiple organ engraftment by bone marrow-derived myofibroblasts and fibroblasts in bone marrow transplanted mice. Stem Cells. Eichmann, A., Pardanaud, L., Yuan, L., & Moyon, D. (2002). Vasculogenesis and the search for the hemangioblast. Journal of Hematotherpy and Stem Cell Research, 11(2), 207–214.

596

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597

Epperly, M. W., Guo, H., Gretton, J. E., & Greenberger, J. S. (2003). Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. American Journal of Respiraory Cell and Molecular Biology, 29(2), 213–224. Erices, A., Conget, P., & Minguell, J. J. (2000). Mesenchymal progenitor cells in human umbilical cord blood. British Journal of Haematology, 109(1), 235–242. Fernandez, M., Simon, V., Herrera, G., Cao, C., Del Favero, H., & Minguell, J. J. (1997). Detection of stromal cells in peripheral blood progenitor cell collections from breast cancer patients. Bone Marrow Transplantation, 20(4), 265–271. Ferrari, G., Cusella-De Angelis, G., Coletta, M., Paolucci, E., Stornaiuolo, A., Cossu, G., & Mavilio, F. (1998). Muscle regeneration by bone marrow-derived myogenic progenitors. Science, 279(5356), 1528–1530. Friedenstein, A. J., Gorskaja, J. F., & Kulagina, N. N. (1976). Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Experimental Hematology, 4(5), 267– 274. Grant, M. B., May, W. S., Caballero, S., Brown, G. A., Guthrie, S. M., Mames, R. N., Byrne, B. J., Vaught, T., Spoerri, P. E., Peck, A. B., & Scott, E. W. (2002). Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Natural Medicine, 8(6), 607–612. Grimm, P. C., Nickerson, P., Jeffery, J., Savani, R. C., Gough, J., McKenna, R. M., Stern, E., & Rush, D. N. (2001). Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renal-allograft rejection. New England Journal of Medicine, 345(2), 93–97. Gronthos, S., Zannettino, A. C., Hay, S. J., Shi, S., Graves, S. E., Kortesidis, A., & Simmons, P. J. (2003). Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. Journal of Cell Science, 116(9), 1827–1835. Guerriero, A., Worford, L., Holland, H. K., Guo, G. R., Sheehan, K., & Waller, E. K. (1997). Thrombopoietin is synthesized by bone marrow stromal cells. Blood, 90(9), 3444–3455. Gunsilius, E. (2003). Evidence from a leukemia model for maintenance of vascular endothelium by bone-marrow-derived endothelial cells. Advances in Experimental Medicine and Biology, 522, 17–24. Guo, H., Fang, B., Liao, L., Zhao, Z., Liu, J., Chen, H., Hsu, S. H., Cui, Q., & Zhao, R. C. (2003). Hemangioblastic characteristics of fetal bone marrow-derived Flk1(+)CD31(−)CD34(−) cells. Experimental Hematology, 31(7), 650–658. Gussoni, E., Bennett, R. R., Muskiewicz, K. R., Meyerrose, T., Nolta, J. A., Gilgoff, I., Stein, J., Chan, Y. M., Lidov, H. G., Bonnemann, C. G., Von Moers, A., Morris, G. E., Den Dunnen, J. T., Chamberlain, J. S., Kunkel, L. M., & Weinberg, K. (2002). Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation. Journal of Clinical Investigation, 110(6), 807– 814. Hirschi, K., & Goodell, M. (2001). Common origins of blood and blood vessels in adults? Differentiation, 68(4–5), 186–192. Horwitz, E. M., Prockop, D. J., Fitzpatrick, L. A., Koo, W. W., Gordon, P. L., Neel, M., Sussman, M., Orchard, P., Marx, J. C.,

Pyeritz, R. E., & Brenner, M. K. (1999). Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Natural Medicine, 5(3), 309–313. Huss, R., Lange, C., Weissinger, E. M., Kolb, H. J., & Thalmeier, K. (2000). Evidence of peripheral blood-derived, plastic-adherent CD34(−/low) hematopoietic stem cell clones with mesenchymal stem cell characteristics. Stem Cells, 18(4), 252–260. Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E., Keene, C. D., Ortiz-Gonzalez, X. R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W. C., Largaespada, D. A., & Verfaillie, C. M. (2002a). Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 418(6893), 41–49. Jiang, Y., Vaessen, B., Lenvik, T., Blackstad, M., Reyes, M., & Verfaillie, C. M. (2002b). Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Experimental Hematology, 30(8), 896–904. Koc, O. N., Gerson, S. L., Cooper, B. W., Dyhouse, S. M., Haynesworth, S. E., Caplan, A. I., & Lazarus, H. M. (2000). 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. Journal of Clinical Oncology, 18(2), 307–316. Kuramochi, Y., Fukazawa, R., Migita, M., Hayakawa, J., Hayashida, M., Uchikoba, Y., Fukumi, D., Shimada, T., & Ogawa, S. (2003). Cardiomyocyte regeneration from circulating bone marrow cells in mice. Pediatric Research. Kuznetsov, S. A., Mankani, M. H., Gronthos, S., Satomura, K., Bianco, P., & Robey, P. G. (2001). Circulating skeletal stem cells. Journal of Cell Biology, 153(5), 1133–1140. Laflamme, M. A., Myerson, D., Saffitz, J. E., & Murry, C. E. (2002). Evidence for cardiomyocyte repopulation by extracardiac progenitors in transplanted human hearts. Circulation Research, 90(6), 634–640. Lagaaij, E. L., Cramer-Knijnenburg, G. F., van Kemenade, F. J., van Es, L. A., Bruijn, J. A., & van Krieken, J. H. (2001). Endothelial cell chimerism after renal transplantation and vascular rejection. Lancet, 357(9249), 33–37. Lazarus, H. M., Haynesworth, S. E., Gerson, S. L., Rosenthal, N. S., & Caplan, A. I. (1995). Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): Implications for therapeutic use. Bone Marrow Transplantation, 16(4), 557–564. Lazarus, H. M., Haynesworth, S. E., Gerson, S. L., & Caplan, A. I. (1997). Human bone marrow-derived mesenchymal (stromal) progenitor cells (MPCs) cannot be recovered from peripheral blood progenitor cell collections. Journal of Hematotherapy, 6(5), 447–455. Lecanda, F., Avioli, L. V., & Cheng, S. L. (1997). Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2. Journal of Cell Biochemistry, 67(3), 386–396. Liechty, K. W., MacKenzie, T. C., Shaaban, A. F., Radu, A., Moseley, A. M., Deans, R., Marshak, D. R., & Flake,

C.A. Roufosse et al. / The International Journal of Biochemistry & Cell Biology 36 (2004) 585–597 A. W. (2000). Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Natural Medicine, 6(11), 1282– 1286. Loncar, D. (1992). Ultrastructural analysis of differentiation of rat endoderm in vitro. Adipose vascular-stromal cells induce endoderm differentiation, which in turn induces differentiation of the vascular-stromal cells into chondrocytes. Journal of Submicroscopic Cytology and Pathology, 24(4), 509–519. Minguell, J. J., Erices, A., & Conget, P. (2001). Mesenchymal stem cells. Experimental and Biological Medicine (Maywood), 226(6), 507–520. Moreau, I., Duvert, V., Caux, C., Galmiche, M. C., Charbord, P., Banchereau, J., & Saeland, S. (1993). Myofibroblastic stromal cells isolated from human bone marrow induce the proliferation of both early myeloid and B-lymphoid cells. Blood, 82(8), 2396–2405. Nuttall, M. E., Patton, A. J., Olivera, D. L., Nadeau, D. P., & Gowen, M. (1998). Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: Implications for osteopenic disorders. Journal of Bone and Mineral Research, 13(3), 371–382. Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S. M., Li, B., Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, D. M., Leri, A., & Anversa, P. (2001). Bone marrow cells regenerate infarcted myocardium. Nature, 410(6829), 701– 705. Pereira, R. F., Halford, K. W., O’Hara, M. D., Leeper, D. B., Sokolov, B. P., Pollard, M. D., Bagasra, O., & Prockop, D. J. (1995). Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proceedings of the National Academic Science of United States of America, 92(11), 4857–4861. Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., & Marshak, D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science, 284(5411), 143–147. Poulsom, R., Forbes, S. J., Hodivala-Dilke, K., Ryan, E., Wyles, S., Navaratnarasah, S., Jeffery, R., Hunt, T., Alison, M., Cook, T., Pusey, C., & Wright, N. A. (2001). Bone marrow contributes to renal parenchymal turnover and regeneration. Journal of Pathology, 195(2), 229–235. Prockop, D. J. (1997). Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 276(5309), 71–74. Quaini, F., Urbanek, K., Beltrami, A. P., Finato, N., Beltrami, C. A., Nadal-Ginard, B., Kajstura, J., Leri, A., & Anversa, P. (2002). Chimerism of the transplanted heart. New England Journal of Medicine, 346(1), 5–15.

597

Reyes, M., Lund, T., Lenvik, T., Aguiar, D., Koodie, L., & Verfaillie, C. M. (2001). Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood, 98(9), 2615–2625. Sainteny, F. (2001). Production of hematopoietic cells from ES cells. Journal of Society of Biology, 195(1), 13–18. Simmons, P. J., & Torok-Storb, B. (1991). Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood, 78(1), 55–62. Studeny, M., Marini, F. C., Champlin, R. E., Zompetta, C., Fidler, I. J., & Andreeff, M. (2002). Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Research, 62(13), 3603–3608. Sullivan, D. C., & Bicknell, R. (2003). New molecular pathways in angiogenesis. British Journal of Cancer, 89(2), 228– 231. Tavian, M., Hallais, M. F., & Peault, B. (1999). Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo. Development, 126(4), 793–803. Toma, C., Pittenger, M. F., Cahill, K. S., Byrne, B. J., & Kessler, P. D. (2002). Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation, 105(1), 93–98. Verfaillie, C. M. (2002). Adult stem cells: Assessing the case for pluripotency. Trends in Cell Biology, 12(11), 502–508. Wells, W. A. (2002). Is transdifferentiation in trouble? Journal of Cell Biology, 157(1), 15–18. Wexler, S. A., Donaldson, C., Denning-Kendall, P., Rice, C., Bradley, B., & Hows, J. M. (2003). Adult bone marrow is a rich source of human mesenchymal ‘stem’ cells but umbilical cord and mobilized adult blood are not. British Journal of Haematology, 121(2), 368–374. Williams, J. T., Southerland, S. S., Souza, J., Calcutt, A. F., & Cartledge, R. G. (1999). Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes. American Surgery, 65(1), 22–26. Wu, G. D., Nolta, J. A., Jin, Y. S., Barr, M. L., Yu, H., Starnes, V. A., & Cramer, D. V. (2003). Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation, 75(5), 679–685. Yang, L., Scott, P. G., Giuffre, J., Shankowsky, H. A., Ghahary, A., & Tredget, E. E. (2002). Peripheral blood fibrocytes from burn patients: Identification and quantification of fibrocytes in adherent cells cultured from peripheral blood mononuclear cells. Laboratory Investigation, 82(9), 1183–1192. Zvaifler, N. J., Marinova-Mutafchieva, L., Adams, G., Edwards, C. J., Moss, J., Burger, J. A., & Maini, R. N. (2000). Mesenchymal precursor cells in the blood of normal individuals. Arthritis Research, 2(6), 477–488.