Mesenchymal Stem Cells Derived from CD133-Positive Cells in Mobilized Peripheral Blood and Cord Blood: Proliferation, Oct4 Expression, and P
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《干细胞学杂志》
Jules Bordet Institute-ULB, Brussels, Belgium
Key Words. Umbilical cord blood ? Peripheral blood ? Mesenchymal stem cells
Correspondence: Tatiana Tondreau, Jules Bordet Institute, 121 Bd de Waterloo, 1000 Brussels, Belgium. Telephone: 32-2-5413724; Fax: 32-2-5413453; e-mail: tatiana.tondreau@bordet.be
ABSTRACT
It has been shown in recent years that human bone marrow (BM) contains hematopoietic stem cells, responsible for the hematopoietic turnover, and mesenchymal stem cells (MSCs), giving to these hematopoietic stem cells an appropriate microenvironment . MSCs are defined as adult immature cells capable of self-renewing and of differentiating into various tissues in vivo and in vitro . After being transplanted into a mouse model or in utero into sheep, MSCs were then engrafted in different tissues including bone, muscle, brain, lung, heart, and liver tissue, without inflammatory response, thanks to the absence of HLA class II expression. Moreover, experiments have proven that MSCs may decrease graft-versus-host disease (GVHD) by inactivating and inhibiting the proliferation of T lymphocyte . Recent studies have also demonstrated clinical applications of MSCs to improve hematopoietic engraftment, to prevent GVHD, and to correct genetic disorders such as osteogenesis imperfecta . There is also a great interest in the potential use of MSCs in molecular therapy to deliver oncolytic viruses . BM is the major source of MSCs, but this population is rare (0.001%–0.01%). Recently, MSCs have also been isolated from adipose tissues, muscle, fetal organs, brain, and teeth . Different techniques can be used to obtain MSCs from the BM: plastic adhesion and negative (CD45, Gly-A, and RosetteSep) or positive selection (CD49-a, Stro-1, and CD133) . However, their presence in mobilized peripheral blood (MPB) or in umbilical cord blood (UCB) still remains controversial. A few authors have demonstrated the presence of stromal or mesenchymal progenitor cells in peripheral blood or in the growth factor-mobilized apheresis products . In their experiments, the expanded adherent cells have shown characteristics similar to those of BM-MSCs. In these studies, MSCs were isolated by the classic plastic adhesion method and subcultures. Using the same procedure, other groups have attempted to isolate MSCs from peripheral blood without success . Plastic adhesion and negative immunodepletion have been suggested to isolate MSCs, but it has been reported that only midtrimester and no full-term cord blood may contain MSCs . In the present study, we assessed the presence of MSCs in MPB and in full-term UCB in comparison with BM. We compared the classic plastic adhesion method and subcultures with cultures containing BM-MSC conditioned medium (BM-MSC CM) and finally with CD133+ initiated cells. We observed that in these three different culture systems, MSCs can easily be isolated and expanded from MPB and UCB. The CD133-positive fraction contains more MSCs with high proliferative potential. MSCs isolated from MPB and UCB show the characteristic pattern of mesenchymal surface markers and express Oct4 (Octomer-binding Transcription Factor 4), a marker of pluripotent stem cells . We also demonstrated their potential for multilineage differentiation, because these cells can become bone, cartilage, fat tissue, and neuronal/glial cells under specific induction media.
MATERIALS AND METHODS
Characterization of MPB- and UCB-MSCs
The first method described to isolate BM-MSCs was based on their strong capacity to adhere to plastic culture flasks . Cells obtained in this condition enable us after successive passages to generate a homogeneous population of MSCs capable of differentiating in several tissues (e.g., adipocytes, osteocytes, chondrocytes, neurons, and myocytes). In this study, we assessed the presence of MSCs in MPB and in UCB. In spite of many studies, their presence in both these sources is still controversial. We tested different culture procedures to evaluate the presence of MSCs in MPB and UCB in comparison with BM. MPB- and UCB-MSCs were isolated either by classic adhesion method or after CD133 cell selection. Because MSCs are capable of secreting cytokines and growth factors essential for their proliferation and growth and because they are sparse in UCB and MPB, we postulated that BM-MSC CM could be helpful to accelerate the proliferation of MSCs in these sources. During the first 48 hours of culture, we incubated MPB or UCB mononucleated cells in the presence of 5% of BM-MSC CM.
After CD133 selection, the cell fraction seeded in PM was composed of 90.4% ± 1.03% of CD133+ cells (data not shown). More than 89.5% ± 2.4% of CD133+ cells coexpressed the CD34 antigen, a hematopoietic progenitor marker (Fig. 1).
Figure 1. A representative fluorescence-activated cell sorting analysis of isolated CD133+ cells unlabeled (A) and comarked with CD34/ CD133 antibodies (B). Numbers indicate the percentage of CD133+ cells coexpressing, or not coexpressing, the CD34 antigen. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin.
During a 15-day PM, the majority of adherent cells observed in MPB or UCB, whatever the method used, were comprised of hematopoietic cells, monocytes/macrophages, endothelial cells, and osteoclasts. The addition of CM during the first 48 hours of PM generated more nucleated cells, but similar cell composition was observed. After the second passage, adherent cells were constituted by a homogeneous population of MSCs. Through fluorescence-activated cell sorting analysis, we demonstrated that the profile of expression of UCB- and MPB-MSCs was similar to BM-MSCs. Cells were positive for SH2, SH3, CD105, and CD44, but negative for CD14, CD34, CD45, and HLA-DR (Fig. 2).
Figure 2. Immunophenotyping of mononuclear cell fraction (white histograms) and UCB-MSCs obtained after two passages (gray histograms). Cells were labeled with fluorescein isothiocyanate– and phycoerythrin-conjugated antibody and evaluated by flow cytometry. After two passages, MSCs were positive for SH2, CD105, SH3, and CD44, but negative for CD14, CD34, CD45, and HLA-DR. Abbreviations: MSC, mesenchymal stem cell; UCB, umbilical cord blood.
UCB- and MPB-MSC Plasticity
Another characteristic of MSCs is their capacity to differentiate in vitro into different tissues after specific induction. In this study, we evaluated the potential of MSCs derived from MPB or UCB to differentiate into adipocytes, osteocytes, chondrocytes, and neuronal cells. Two weeks after mesodermal induction (adipogenic, osteogenic, and chondrogenic media), we assessed lipid vacuoles, calcium deposits, and chondrogenic matrix using Oil Red O, Von Kossa, and Toluidin blue, respectively (Figs. 3A–3D). Ten days after neurogenic induction, cells displayed typical neuron-like morphology and were positive for Nestin, Tuj-1, MAP-2, and GFAP (Figs. 3E–3G).
Figure 3. Differentiation potential of MSCs obtained from full-term UCB after P2 (A) and cultured under specific induction: (B) osteocytes (calcium deposits colored by the Von Kossa method), (C) adipocytes (lipid vacuoles stained by Oil red O), and (D) chondrocytes (chondrogenic matrix evidenced by toluidin blue). (E): Morphology of MSCs after incubation in neurogenic medium; cells present long, thin process and round cell bodies. (F): Reverse transcription–polymerase chain reaction analysis of nestin and GFAP expression in MNCs, neural/glial cells (normal human neural progenitor cell ; Cambrex Bio Science), and neurons derived from MPB-MSCs. (G): Expression of Tuj-1 and MAP-2 10 days post–neural induction on differentiated MSCs. Control represents differentiated cells incubated with FITC-conjugated secondary antibody. Abbreviations: FITC, fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; MAP-2, microtubule associated protein 2; MNC, mononuclear cell; MPB, mobilized peripheral blood; MSC, mesenchymal stem cell; UCB, umbilical cord blood.
Colony-Forming Unit-Fibroblast Potential Study
MSCs are characterized by their ability to form colonies comprising nonrefringent spindle-shaped cells deriving from a single cell (colony-forming unit-fibroblast assay). This assay was used to evaluate the cloning efficiency (number of CFU-F/5,000 seeded cells) of MSCs obtained from BM, MPB, and UCB. No colony was observed in MPB- and in UCB-MNCs. However, we obtained few fibroblastic colonies in the case of BM MNCs (3.5 ± 1.5 CFU-F). This last result confirms the very low frequency of MSCs in MPB and UCB. After the PM of MPB and UCB, CFU-F colonies appeared and we counted them. Interestingly, we observed that a great number of CFU-F can be obtained when CD133+ cell selection was performed, demonstrating that CD133-positive cell fraction is rich in MSC clonogenic precursors. When CM was added during the first 48 hours of culture, we also observed a slight increase in the number of CFU-F. After two passages (P2), whatever the method used to isolate MSCs from MPB and UCB, a similar level of CFU-F was observed (Fig. 4).
Figure 4. Representative CFU-F profile of cells obtained from full-term UCB after PM and two passages (P2), cultured under different conditions: (A) cells cultured in complete alpha–modified Eagle’s medium, (B) cells cultured in the presence of 5% CM, and (C) CD133+ cell selection. Cells were seeded at a concentration of 5,000 cells per dish. Ten days later, CFU-F were counted. Abbreviations: CFU-F, colony-forming units-fibroblast; CM, conditioned medium; PM, primoculture; UCB, umbilical cord blood.
Cell Expansion
To evaluate the expansion of MSCs derived from UCB and MPB, we calculated the number of cells seeded in primary culture to 1 x 105 cells per cm2 for each cell culture condition. Generally, MSC cultures were initiated from 20 ml BM aspirates and, in the case of UCB, from samples of more than 50 ml of blood. A large volume of blood and a great quantity of MNCs can be collected from MPB (Table 1). In these sources, MSCs are sparse, and to increase the probability of obtaining MSCs, more cells were seeded in PM. Indeed, 1 x 106 MNCs per cm2 were required to initiate the culture from UCB and MPB, versus 1 x 104 cells per cm2 for BM. The growth curves of MSCs from the three sources can be observed in Figure 5. For each passage, we seeded 100 cells per cm2 and made comparisons with the starting fraction. We observed a great variability between BM samples (BM1 versus BM2). BM1 contained more MSCs than BM2, but BM1 corresponds to particulate fraction removed by nylon filtration prior to transplantation, which is rich in hematopoietic and stromal cells . BM2 is obtained though classic sternal aspiration. The culture of CD133+ cells from UCB and MPB enabled us to obtain a great quantity of MSCs in comparison with the culture of MNCs in complete -MEM supplemented, or not supplemented, with 5% CM (Figs. 5A, 5B). After fourth passages (P4), 6.8 x 108 cells and 3.3 x 107 cells were obtained from UCB and MPB, respectively, from the CD133+ cell fraction. Using plastic adhesion to expand BM MSCs, 3.8 x 106 to 7.9 x 109 cells were obtained from BM2 and BM1. These results demonstrated that the CD133+ cell fraction contains more MSCs with high proliferative potential. The decrease of cell number observed between PM and P1 culture is associated with the elimination of hematopoietic cells from the culture.
Table 1. Characteristics of samples
Figure 5. The representative cumulative population expansion of (A) UCB and (B) MPB cells was compared with BM-MSCs with high (BM1) or low (BM2) proliferate potential. Using the CD133 cell fraction, a similar number of MSC was obtained in comparison with BM-MSCs after four passages. Cultures in complete alpha–modified Eagle’s medium or supplemented with 5% CM were not sufficient to expand significantly UCB- and MPB-MSCs. Abbreviations: BM, bone marrow; CM, cultured medium; MPB, mobilized peripheral blood; MSC, mesenchymal stem cell; UCB, umbilical cord blood.
RT-PCR Analysis of MPB- and UCB-MSCs
Oct4, the octomer-binding transcription factor 4, is an important binding transcription factor present in undifferentiated embryonic stem cells with a high proliferative capacity, but also detected at a lower level in multipotent adult progenitor cells (MAPCs) . Pochampally et al. recently selected a subpopulation of early progenitor cells in a serum-deprived culture of human marrow stromal cells with increased expression of Oct4. In this study, we evaluated Oct4 expression in MNCs and MSCs obtained from BM, MPB, and UCB (Fig. 6). All MSCs isolated by plastic adhesion method were assessed after the second passage, when more than 90% of cells express mesenchymal markers (SH2, SH3) but not hematopoietic markers (CD14, CD34, CD45), and these MSCs are multipotent. We observed that MNCs have no transcript for Oct4, whereas MSCs derived from all three sources (BM, MPB, and UCB) express Oct4. This result confirms the presence of MSCs with high proliferative capacity in MPB and in full-term UCB.
Figure 6. Oct4 expression in MSCs derived from UCB and MPB after the second passage and cultured in complete alpha–modified Eagle’s medium (Ctrl) in the presence of CM during the first 48 hours of PM (CM) or after CD133 selection (CD133). GAPDH is a reverse transcription–polymerase chain reaction internal control. Abbreviations: CM, conditioned medium; MPB, mobilized peripheral blood; MSC, mesenchymal stem cell; PM, primoculture; UCB, umbilical cord blood.
DISCUSSION
MPB and UCB are important sources of hematopoietic stem cells already used in cell therapy after chemotherapy. Due to the large volume and accessibility of these sources, it was important to find a common, easily reproducible method for purifying MSCs. In this study, we demonstrated that MSCs can be easily isolated from MPB or UCB, using a CD133-positive selection. This method of selection makes it possible after four passages to rapidly obtain a large quantity of MSCs, as described for BM. These MPB- and UCB-MSCs have a potential similar to MSCs derived from BM, such as differentiation into different tissues (fat, bone, cartilage, and neural cells) and proliferative capacity (CFU-F assay, cumulative cell number, and Oct4 expression). This interesting finding demonstrates the presence of "circulating" MSCs in MPB and in UCB and the possibility of isolating them with a common method based on the CD133-positive selection. We therefore suggest that UCB and MPB could be considered not only as a source of hematopoietic cells but also as a source of MSCs for cellular therapy.
ACKNOWLEDGMENTS
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Key Words. Umbilical cord blood ? Peripheral blood ? Mesenchymal stem cells
Correspondence: Tatiana Tondreau, Jules Bordet Institute, 121 Bd de Waterloo, 1000 Brussels, Belgium. Telephone: 32-2-5413724; Fax: 32-2-5413453; e-mail: tatiana.tondreau@bordet.be
ABSTRACT
It has been shown in recent years that human bone marrow (BM) contains hematopoietic stem cells, responsible for the hematopoietic turnover, and mesenchymal stem cells (MSCs), giving to these hematopoietic stem cells an appropriate microenvironment . MSCs are defined as adult immature cells capable of self-renewing and of differentiating into various tissues in vivo and in vitro . After being transplanted into a mouse model or in utero into sheep, MSCs were then engrafted in different tissues including bone, muscle, brain, lung, heart, and liver tissue, without inflammatory response, thanks to the absence of HLA class II expression. Moreover, experiments have proven that MSCs may decrease graft-versus-host disease (GVHD) by inactivating and inhibiting the proliferation of T lymphocyte . Recent studies have also demonstrated clinical applications of MSCs to improve hematopoietic engraftment, to prevent GVHD, and to correct genetic disorders such as osteogenesis imperfecta . There is also a great interest in the potential use of MSCs in molecular therapy to deliver oncolytic viruses . BM is the major source of MSCs, but this population is rare (0.001%–0.01%). Recently, MSCs have also been isolated from adipose tissues, muscle, fetal organs, brain, and teeth . Different techniques can be used to obtain MSCs from the BM: plastic adhesion and negative (CD45, Gly-A, and RosetteSep) or positive selection (CD49-a, Stro-1, and CD133) . However, their presence in mobilized peripheral blood (MPB) or in umbilical cord blood (UCB) still remains controversial. A few authors have demonstrated the presence of stromal or mesenchymal progenitor cells in peripheral blood or in the growth factor-mobilized apheresis products . In their experiments, the expanded adherent cells have shown characteristics similar to those of BM-MSCs. In these studies, MSCs were isolated by the classic plastic adhesion method and subcultures. Using the same procedure, other groups have attempted to isolate MSCs from peripheral blood without success . Plastic adhesion and negative immunodepletion have been suggested to isolate MSCs, but it has been reported that only midtrimester and no full-term cord blood may contain MSCs . In the present study, we assessed the presence of MSCs in MPB and in full-term UCB in comparison with BM. We compared the classic plastic adhesion method and subcultures with cultures containing BM-MSC conditioned medium (BM-MSC CM) and finally with CD133+ initiated cells. We observed that in these three different culture systems, MSCs can easily be isolated and expanded from MPB and UCB. The CD133-positive fraction contains more MSCs with high proliferative potential. MSCs isolated from MPB and UCB show the characteristic pattern of mesenchymal surface markers and express Oct4 (Octomer-binding Transcription Factor 4), a marker of pluripotent stem cells . We also demonstrated their potential for multilineage differentiation, because these cells can become bone, cartilage, fat tissue, and neuronal/glial cells under specific induction media.
MATERIALS AND METHODS
Characterization of MPB- and UCB-MSCs
The first method described to isolate BM-MSCs was based on their strong capacity to adhere to plastic culture flasks . Cells obtained in this condition enable us after successive passages to generate a homogeneous population of MSCs capable of differentiating in several tissues (e.g., adipocytes, osteocytes, chondrocytes, neurons, and myocytes). In this study, we assessed the presence of MSCs in MPB and in UCB. In spite of many studies, their presence in both these sources is still controversial. We tested different culture procedures to evaluate the presence of MSCs in MPB and UCB in comparison with BM. MPB- and UCB-MSCs were isolated either by classic adhesion method or after CD133 cell selection. Because MSCs are capable of secreting cytokines and growth factors essential for their proliferation and growth and because they are sparse in UCB and MPB, we postulated that BM-MSC CM could be helpful to accelerate the proliferation of MSCs in these sources. During the first 48 hours of culture, we incubated MPB or UCB mononucleated cells in the presence of 5% of BM-MSC CM.
After CD133 selection, the cell fraction seeded in PM was composed of 90.4% ± 1.03% of CD133+ cells (data not shown). More than 89.5% ± 2.4% of CD133+ cells coexpressed the CD34 antigen, a hematopoietic progenitor marker (Fig. 1).
Figure 1. A representative fluorescence-activated cell sorting analysis of isolated CD133+ cells unlabeled (A) and comarked with CD34/ CD133 antibodies (B). Numbers indicate the percentage of CD133+ cells coexpressing, or not coexpressing, the CD34 antigen. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin.
During a 15-day PM, the majority of adherent cells observed in MPB or UCB, whatever the method used, were comprised of hematopoietic cells, monocytes/macrophages, endothelial cells, and osteoclasts. The addition of CM during the first 48 hours of PM generated more nucleated cells, but similar cell composition was observed. After the second passage, adherent cells were constituted by a homogeneous population of MSCs. Through fluorescence-activated cell sorting analysis, we demonstrated that the profile of expression of UCB- and MPB-MSCs was similar to BM-MSCs. Cells were positive for SH2, SH3, CD105, and CD44, but negative for CD14, CD34, CD45, and HLA-DR (Fig. 2).
Figure 2. Immunophenotyping of mononuclear cell fraction (white histograms) and UCB-MSCs obtained after two passages (gray histograms). Cells were labeled with fluorescein isothiocyanate– and phycoerythrin-conjugated antibody and evaluated by flow cytometry. After two passages, MSCs were positive for SH2, CD105, SH3, and CD44, but negative for CD14, CD34, CD45, and HLA-DR. Abbreviations: MSC, mesenchymal stem cell; UCB, umbilical cord blood.
UCB- and MPB-MSC Plasticity
Another characteristic of MSCs is their capacity to differentiate in vitro into different tissues after specific induction. In this study, we evaluated the potential of MSCs derived from MPB or UCB to differentiate into adipocytes, osteocytes, chondrocytes, and neuronal cells. Two weeks after mesodermal induction (adipogenic, osteogenic, and chondrogenic media), we assessed lipid vacuoles, calcium deposits, and chondrogenic matrix using Oil Red O, Von Kossa, and Toluidin blue, respectively (Figs. 3A–3D). Ten days after neurogenic induction, cells displayed typical neuron-like morphology and were positive for Nestin, Tuj-1, MAP-2, and GFAP (Figs. 3E–3G).
Figure 3. Differentiation potential of MSCs obtained from full-term UCB after P2 (A) and cultured under specific induction: (B) osteocytes (calcium deposits colored by the Von Kossa method), (C) adipocytes (lipid vacuoles stained by Oil red O), and (D) chondrocytes (chondrogenic matrix evidenced by toluidin blue). (E): Morphology of MSCs after incubation in neurogenic medium; cells present long, thin process and round cell bodies. (F): Reverse transcription–polymerase chain reaction analysis of nestin and GFAP expression in MNCs, neural/glial cells (normal human neural progenitor cell ; Cambrex Bio Science), and neurons derived from MPB-MSCs. (G): Expression of Tuj-1 and MAP-2 10 days post–neural induction on differentiated MSCs. Control represents differentiated cells incubated with FITC-conjugated secondary antibody. Abbreviations: FITC, fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; MAP-2, microtubule associated protein 2; MNC, mononuclear cell; MPB, mobilized peripheral blood; MSC, mesenchymal stem cell; UCB, umbilical cord blood.
Colony-Forming Unit-Fibroblast Potential Study
MSCs are characterized by their ability to form colonies comprising nonrefringent spindle-shaped cells deriving from a single cell (colony-forming unit-fibroblast assay). This assay was used to evaluate the cloning efficiency (number of CFU-F/5,000 seeded cells) of MSCs obtained from BM, MPB, and UCB. No colony was observed in MPB- and in UCB-MNCs. However, we obtained few fibroblastic colonies in the case of BM MNCs (3.5 ± 1.5 CFU-F). This last result confirms the very low frequency of MSCs in MPB and UCB. After the PM of MPB and UCB, CFU-F colonies appeared and we counted them. Interestingly, we observed that a great number of CFU-F can be obtained when CD133+ cell selection was performed, demonstrating that CD133-positive cell fraction is rich in MSC clonogenic precursors. When CM was added during the first 48 hours of culture, we also observed a slight increase in the number of CFU-F. After two passages (P2), whatever the method used to isolate MSCs from MPB and UCB, a similar level of CFU-F was observed (Fig. 4).
Figure 4. Representative CFU-F profile of cells obtained from full-term UCB after PM and two passages (P2), cultured under different conditions: (A) cells cultured in complete alpha–modified Eagle’s medium, (B) cells cultured in the presence of 5% CM, and (C) CD133+ cell selection. Cells were seeded at a concentration of 5,000 cells per dish. Ten days later, CFU-F were counted. Abbreviations: CFU-F, colony-forming units-fibroblast; CM, conditioned medium; PM, primoculture; UCB, umbilical cord blood.
Cell Expansion
To evaluate the expansion of MSCs derived from UCB and MPB, we calculated the number of cells seeded in primary culture to 1 x 105 cells per cm2 for each cell culture condition. Generally, MSC cultures were initiated from 20 ml BM aspirates and, in the case of UCB, from samples of more than 50 ml of blood. A large volume of blood and a great quantity of MNCs can be collected from MPB (Table 1). In these sources, MSCs are sparse, and to increase the probability of obtaining MSCs, more cells were seeded in PM. Indeed, 1 x 106 MNCs per cm2 were required to initiate the culture from UCB and MPB, versus 1 x 104 cells per cm2 for BM. The growth curves of MSCs from the three sources can be observed in Figure 5. For each passage, we seeded 100 cells per cm2 and made comparisons with the starting fraction. We observed a great variability between BM samples (BM1 versus BM2). BM1 contained more MSCs than BM2, but BM1 corresponds to particulate fraction removed by nylon filtration prior to transplantation, which is rich in hematopoietic and stromal cells . BM2 is obtained though classic sternal aspiration. The culture of CD133+ cells from UCB and MPB enabled us to obtain a great quantity of MSCs in comparison with the culture of MNCs in complete -MEM supplemented, or not supplemented, with 5% CM (Figs. 5A, 5B). After fourth passages (P4), 6.8 x 108 cells and 3.3 x 107 cells were obtained from UCB and MPB, respectively, from the CD133+ cell fraction. Using plastic adhesion to expand BM MSCs, 3.8 x 106 to 7.9 x 109 cells were obtained from BM2 and BM1. These results demonstrated that the CD133+ cell fraction contains more MSCs with high proliferative potential. The decrease of cell number observed between PM and P1 culture is associated with the elimination of hematopoietic cells from the culture.
Table 1. Characteristics of samples
Figure 5. The representative cumulative population expansion of (A) UCB and (B) MPB cells was compared with BM-MSCs with high (BM1) or low (BM2) proliferate potential. Using the CD133 cell fraction, a similar number of MSC was obtained in comparison with BM-MSCs after four passages. Cultures in complete alpha–modified Eagle’s medium or supplemented with 5% CM were not sufficient to expand significantly UCB- and MPB-MSCs. Abbreviations: BM, bone marrow; CM, cultured medium; MPB, mobilized peripheral blood; MSC, mesenchymal stem cell; UCB, umbilical cord blood.
RT-PCR Analysis of MPB- and UCB-MSCs
Oct4, the octomer-binding transcription factor 4, is an important binding transcription factor present in undifferentiated embryonic stem cells with a high proliferative capacity, but also detected at a lower level in multipotent adult progenitor cells (MAPCs) . Pochampally et al. recently selected a subpopulation of early progenitor cells in a serum-deprived culture of human marrow stromal cells with increased expression of Oct4. In this study, we evaluated Oct4 expression in MNCs and MSCs obtained from BM, MPB, and UCB (Fig. 6). All MSCs isolated by plastic adhesion method were assessed after the second passage, when more than 90% of cells express mesenchymal markers (SH2, SH3) but not hematopoietic markers (CD14, CD34, CD45), and these MSCs are multipotent. We observed that MNCs have no transcript for Oct4, whereas MSCs derived from all three sources (BM, MPB, and UCB) express Oct4. This result confirms the presence of MSCs with high proliferative capacity in MPB and in full-term UCB.
Figure 6. Oct4 expression in MSCs derived from UCB and MPB after the second passage and cultured in complete alpha–modified Eagle’s medium (Ctrl) in the presence of CM during the first 48 hours of PM (CM) or after CD133 selection (CD133). GAPDH is a reverse transcription–polymerase chain reaction internal control. Abbreviations: CM, conditioned medium; MPB, mobilized peripheral blood; MSC, mesenchymal stem cell; PM, primoculture; UCB, umbilical cord blood.
DISCUSSION
MPB and UCB are important sources of hematopoietic stem cells already used in cell therapy after chemotherapy. Due to the large volume and accessibility of these sources, it was important to find a common, easily reproducible method for purifying MSCs. In this study, we demonstrated that MSCs can be easily isolated from MPB or UCB, using a CD133-positive selection. This method of selection makes it possible after four passages to rapidly obtain a large quantity of MSCs, as described for BM. These MPB- and UCB-MSCs have a potential similar to MSCs derived from BM, such as differentiation into different tissues (fat, bone, cartilage, and neural cells) and proliferative capacity (CFU-F assay, cumulative cell number, and Oct4 expression). This interesting finding demonstrates the presence of "circulating" MSCs in MPB and in UCB and the possibility of isolating them with a common method based on the CD133-positive selection. We therefore suggest that UCB and MPB could be considered not only as a source of hematopoietic cells but also as a source of MSCs for cellular therapy.
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