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Offprint requests to: Catherine M. Verfaillie, M.D., Department of Medicine, University of Minnesota, MMC 716, 422 Delaware Street SE, Minneapolis, MN 55455, USA
Stem Cell Institute, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minn., USADivision of Hematology, Department of Medicine, University of Minnesota Medical School, Minneapolis, Minn., USA
Recent studies have shown that cells from bone marrow (BM), muscle, and brain may have greater plasticity than previously known. We have identified multipotent adult progenitor cells (MAPC) in postnatal human and rodent BM that copurify with mesenchymal stem cells (MSC). BM MAPC proliferate without senescence and differentiate into mesodermal, neuroectodermal, and endodermal cell types. We hypothesized that cells with characteristics similar to BM MAPC can be selected and cultured from tissues other than BM.
Materials and Methods
BM, whole brain, and whole muscle tissue was obtained from mice. Cells were plated on Dulbecco modified Eagle medium supplemented with 2% fetal calf serum and 10 ng/mL epidermal growth factor (EGF), 10 ng/mL platelet-derived growth factor (PDGF-BB), and 1000 units/mL leukemia inhibitory factor (LIF) for more than 6 months. Cells were maintained between 0.5 and 1.5 × 103 cells/cm2. At variable time points, we tested cell phenotype by FACS and evaluated their differentiation into endothelial cells, neuroectodermal cells, and endodermal cells in vitro. We also compared the expressed gene profile in BM, muscle, and brain MAPC by Affimetrix gene array analysis.
Results
Cells could be cultured from BM, muscle, and brain that proliferated for more than 70 population doublings (PDs) and were negative for CD44, CD45, major histocompatibility complex class I and II, and c-kit. Cells from the three tissues differentiated to cells with morphologic and phenotypic characteristics of endothelium, neurons, glia, and hepatocytes. The expressed gene profile of cells derived from the three tissues was identical (r2 > 0.975).
Conclusions
This study shows that cells with MAPC characteristics can be isolated not only from BM, but also from brain and muscle tissue. Whether MAPC originally derived from BM are circulating or all organs contain stem cells with MAPC characteristics currently is being studied. Presence of MAPC in multiple tissues may help explain the “plasticity” found in multiple adult tissues.
Until recently, it was believed that tissue-specific stem cells could only differentiate into cells of the tissue of origin. However, several studies now provide evidence that tissue-specific stem cells may be able to differentiate into cells of different tissues. For instance, cells infused at the time of bone marrow (BM) transplantation contribute to skeletal muscle myoblasts [
]. The mechanism(s) responsible for this apparent plasticity is not known.
We recently showed that a rare cell, which we termed multipotent adult progenitor cells (MAPC), within mesenchymal stem cell (MSC) cultures from human or rodent BM can be expanded for greater than 70 to 150 population doublings (PDs) [
]. This cell differentiates not only into mesenchymal lineage cells but also endothelium, neuroectoderm, and endoderm. In this study, we show that similar MAPC can be selected from mouse muscle and mouse brain.
Methods
Materials
BM was collected from femurs of 3- to 4-week-old 129 × C57BL/6J ROSA26 F1 mice. Muscle was obtained from 3-day-old C57BL/6J mice and brain from 3-day-old 129 mice. All tissues were obtained according to the guidelines from the University of Minnesota IACUC. BM mononuclear cells (MNC) were obtained by Ficoll-Hypaque separation. Muscle from the proximal parts of forelimbs and hindlimbs were excised and thoroughly minced. The tissue was treated with 0.2% collagenase (Sigma Chemical Co., St. Louis, MO, USA) for 1 hour at 37°C, followed by 0.1% trypsin (Invitrogen, Grand Island, NY, USA) for 45 minutes. Cells were triturated vigorously and passed through a 70-μm filter. Cell suspensions were collected and centrifuged for 10 minutes at 400g. Brain tissue was dissected and thoroughly minced. Cells were dissociated by incubation with 0.1% trypsin and 0.1% DNAse (Sigma) for 30 minutes at 37°C. Cells were triturated vigorously and passed through a 70-μm filter. Cell suspension was collected and centrifuged for 10 minutes at 400g.
MAPC and differentiation culture conditions
Media
MAPC expansion medium consisted of the following: 60% DMEM-LG (Gibco BRL, Grand Island, NY, USA), 40% MCDB-201 (Sigma) with 1× insulin-transferrin-selenium (ITS), 1× linoleic-acid-bovine-serum-albumin (LA-BSA), 10−9M dexamethasone (Sigma), 10−4M ascorbic acid 2-phosphate (Sigma), 100 units of penicillin, 1000 units of streptomycin (Gibco), on fibronectin (FN; Sigma) with 2% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT, USA) [
]. Expansion medium contained 10 ng/mL hPDGF-BB (R&D Systems, Minneapolis, MN, USA), 10 ng/mL mEGF (Sigma), and 1000 units/mL mLIF (Chemicon, Temecula, CA, USA). Differentiation medium was similar to expansion medium but without FCS (Hyclone Laboratories), without the expansion cytokines, but with cytokines specific for differentiation as described later.
MAPC cultures
For BM, 1 × 105/cm2 MNC were plated on expansion medium with EGF, PDFG-BB, and LIF on plates coated with 10 ng/mL FN (Sigma), and maintained at 5 × 103/cm2. For muscle and brain, cell suspensions were plated on FN-coated plates at 1 × 105 cells/cm2 in expansion medium with EGF, PDFG-BB, and LIF. After 24 hours, nonadherent debris was removed, and adherent cells were cultured for 3 to 4 weeks, keeping cell at 5 × 103/cm2. After 3 to 4 weeks, cells from cultures initiated with BM, muscle, or brain cells were depleted of CD45+/Ter119+ cells using micromagnetic beads (Miltenyi Biotec, Sunnyvale, CA, USA), and replated at 10 cells per well in FN-coated 96-well plates and expanded at densities between 0.5 and 1.5 × 103/cm2.
Endothelial differentiation
2 × 104 MAPC/cm2 were plated on FN in serum-free medium with 10 ng/mL vascular endothelial growth factor-B (VEGF-B; gift from Dr. Ramakrishna, University of Minnesota) with medium exchanges every 3 days.
Neural differentiation
1 × 104 MAPC/cm2 were plated on FN in serum-free medium with 100 ng/mL basic fibroblast growth factor (bFGF; R&D Systems) with medium exchanges every 3 days.
Endodermal differentiation
2 × 104 MAPC/cm2 were plated on Matrigel (Sigma) in serum-free medium with 10 ng/mL each fibroblast growth factor-4 (FGF4) and hepatocyte growth factor (HGF) (R&D Systems) with medium exchanges every 3 days.
Karyotyping
Cells, subcultured at a 1:2 dilution 12 hours before harvesting, were collected with trypsin-EDTA and subjected to a 1.5-hour Colcemid incubation followed by lysis with hypotonic KCl and fixation in acid/alcohol as previously described [
]. The mRNA was reverse transcribed, and cDNA underwent 40 rounds of amplification (ABI PRISM 7700; Perkin Elmer/Applied Biosystems) with the following reaction conditions: 40 cycles of a two-step polymerase chain reaction (PCR; 95°C for 15 min, 60°C for 60 min) after initial denaturation (95°C for 10 min) with 2μL of DNA solution, 1× TaqMan SYBR Green Universal Mix PCR reaction buffer. Primers for Oct-4 were 5′-GAAGCGTTTCTCCCTGGATT-3′; and 5′-GTGTAGGATTGGGTGCGTT-3′ and for Rex-1 were 5′-GAAGCGTTCTCCCTGGAATTTC-3′; 5′-GTGTAGGATTGGGTGCGTTT-3′. mRNA levels were normalized using GAPDH as housekeeping gene and compared with levels in mouse ES cells.
Immunophenotypic analysis
Facs
For FACS, undifferentiated MAPC were detached and stained sequentially with anti-CD13, CD44, CD45, c-kit, Flk1, and major histocompatibility complex (MHC) class I and II (BD Pharmingen) and secondary phycoerythrin (PE) or fluorescein (FITC) antibodies (Abs), fixed with 2% paraformaldehyde until analysis using FACS Calibur (Becton-Dickinson).
Immunofluorescence
Cultured cells were fixed with 4% paraformaldehyde (Sigma) for 4 minutes at room temperature, followed by methanol (Sigma) for 2 minutes at −20°C. For nuclear ligands, cells were permeabilized with 0.1 Triton X-100 (Sigma) for 10 minutes. Slides were incubated sequentially for 30 minutes each with primary Ab and FITC- or Cy3-coupled anti-mouse, anti-goat, or anti-rabbit immunoglobulin G (IgG) Ab. Between each step, slides were washed with phosphate-buffered saline + 1% BSA (Sigma). Cells were examined by fluorescence microscopy (Zeiss Axiovert; Carl Zeiss, Inc., Thornwood, NY, USA) and confocal fluorescence microscopy (confocal 1024 microscope, Olympus AX70; Olympus Optical Co. Ltd, Tokyo, Japan). To assess the frequency of different cell types in a given culture, we counted the number of cells staining positive with a given Ab in four visual fields (50–200 cells per field). Anti-CD31 Ab was obtained from BD Pharmingen. Ab against glial fibrillary acidic protein was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Dako Corporation (Carpinteria, CA, USA). Abs against neurofilament (NF)-200 (clone N52, 1:400), neuron-specific enolase (NSE; polyclonal, 1:50), Tau (polyclonal, 1:400), cytokeratin (CK)-18 (C-8541; 1:300), and albumin (A-6684; 1:100) were from Sigma. Polyclonal Abs against von Willebrand factor (vWF), HNF-1, and Flk1 were obtained from Santa Cruz Biotechnology. Control mouse, rabbit, or rat IgGs and FITC- or Cy3-labeled secondary Abs were obtained from Sigma.
Oligonucleotide array analysis
In experiment 1, mRNA was extracted from 2 to 3 × 106 BM-, muscle-, or brain-derived MAPC cultured for 45 PDs. In experiment 2, total RNA was obtained from BM MAPC expanded for 85 PDs, and from muscle and brain MAPC expanded for 60 PDs. Preparation of cDNA, hybridization to the U74A array containing 6000 known murine genes and 6000 murine EST clusters, and data acquisition were done per manufacturer's recommendations (all from Affimetrix, Santa Clara, CA, USA). Data analysis was performed using GeneChip software (Affimetrix). Increased or decreased expression by a factor of 2.2-fold [
] was considered significant. The r2 value was determined using linear regression analysis.
Results
Cells with proliferative and phenotypic characteristics of mouse BM MAPC can be cultured from mouse muscle and mouse brain
Murine MAPC can be selected and cultured from BM by plating BM MNC in FN-coated wells with 2% FCS, EGF, PDGF-BB, and LIF. After 3 to 4 weeks, BM MNC are depleted of CD45+ and Ter119+ cells and replated at 10 cells per well. Cells are maintained at densities between 0.5 and 1.5 × 103 cells/cm2 for greater than 100 PDs [
Here we initiated cultures with single-cell suspension of mouse muscle obtained after trypsin/collagenase dissociation of the tissue or from mouse brain obtained after trypsin/DNAse dissociation of the tissue. Single-cell suspensions from brain and muscle were plated on FN, nonadherent debris removed after 24 hours, and cells were maintained at 5 × 103 cells/cm2. After 4 weeks, contaminating CD45+ cells and Ter119+ cells were removed using micromagnetic beads. Ten CD45−Ter119− cells were replated on FN and expanded at densities between 0.5 and 1.5 × 103 cells/cm2. Cells from muscle or brain cultured on FN were 8 to 10 μm in diameter with a large nucleus and scant cytoplasm, similar to the morphology seen for BM MAPC. BM MAPC have been expanded for greater than 100 PDs, and muscle- and brain-derived MAPC for greater than 75 PDs. The growth rate was similar for the three cell sources (Fig. 1). Cytogenetic analysis of muscle- or brain-derived MAPC was performed monthly and showed a normal karyotype. The phenotype of cells derived from brain and muscle cultured on FN for 25 to 60 PDs was identical to the phenotype described for mouse and human BM-derived MAPC: CD13+, Flk1dim, c-kit−, CD44−, CD45−, MHC class I−, and MHC class II− (Fig. 2) [
Figure 1BM-, muscle-, or brain-derived MAPC expansion potential is similar. BM MNC, muscle, or brain cells, depleted of CD45+/Ter119+ cells, were plated at 10 cells per well on FN in expansion medium with 10 ng/mL EGF, PDGF-BB, and 1000 units/mL LIF. Cells were expanded at cell densities between 0.5 and 1.5 × 103 cells/cm2. Cells were enumerated at each passage under hemocytometer.
Figure 2Phenotypes of BM-, muscle-, or brain-derived MAPC are similar. BM MNC, muscle, or brain cells, depleted of CD45+/Ter119+ cells, were plated at 10 cells per well on FN in expansion with 10 ng/mL EGF and PDGF-BB and passaged for 45 PDs. Cells were harvested and labeled with Abs against CD13, Flk1, c-kit, CD44, CD45, MHC class I, MHC class-II, or control IgGs, followed by secondary fluorochrome-coupled Abs, and analyzed by FACS. Plots show isotype control IgG staining profile (black line) vs specific Ab staining profile (red line). Representative example of three experiments.
Cells obtained after ∼45 PDs from BM, muscle, or brain cultures were tested by Q-RT-PCR for expression levels of Oct4 and Rex1. Oct4 and Rex1 mRNA were present in muscle and brain MAPC. As we described for BM MAPC [
], levels of Oct4 mRNA in muscle or brain MAPC were significantly lower than those detected in mouse ES cells, whereas levels of Rex1 mRNA in muscle or brain MAPC were similar to levels detected in mouse ES cells.
Mouse muscle- and brain-derived MAPC differentiate into endothelium, neuroectoderm, and hepatocyte-like cells
Undifferentiated MAPC from either brain, muscle, or BM did not stain with Abs against differentiated cells, including endothelium, neurons, glia-like cells, or hepatocyte-like cells. We tested the differentiation ability of muscle- or brain-derived MAPC using methods described previously for human and mouse BM MAPC. For all differentiations, cells were replated at 1 to 2 × 104 cells/cm2, without FCS, EGF, PDGF-BB, and LIF, but with lineage-specific cytokines.
As an example of mesoderm, we induced differentiation to endothelium. Undifferentiated mouse muscle or brain MAPC did not express the endothelial markers CD31, CD62E, Tek, or vWF, but expressed low levels of Flk1 (Fig. 2 and data not shown). Muscle-, brain-, and BM-derived MAPC were cultured in FN-coated wells with VEGF-B. After treatment with VEGF for 14 days, greater than 90% of MAPC expressed CD31 and vWF (Fig. 3A), which is consistent with endothelial differentiation [
Figure 3BM-, muscle-, or brain-derived MAPC differentiate to endothelium, neuroectoderm, and endoderm in vitro. Mouse BM-, muscle-, or brain-derived MAPC maintained as undifferentiated cells for 60, 45, and 50 PDs were induced to differentiate to endothelium, neuroectoderm, and endoderm as described in the Methods section. After 14 days, cells were fixed, stained with Abs against lineage-specific antigens, and examined by confocal microscopy. Representative example of three experiments. (A) VEGF-treated MAPC stained with an IgG control Ab, anti-vWF, or anti-CD31 Ab followed by a Cy3-coupled secondary Ab. (B) bFGF-treated MAPC stained with an anti-GFAP Ab followed by a secondary Cy3-coupled Ab plus an anti-NF-200 Ab followed by a secondary FITC-coupled Ab, or stained with an anti-Tau Ab followed by a secondary FITC-coupled Ab, or an anti-GFAP Ab followed by a secondary Cy3-coupled Ab. Samples also were stained with an IgG control Ab followed by a secondary FITC- and Cy3-coupled Ab. (C) FGF4- and HGF-treated MAPC stained with an anti-HNF-1 Ab followed by a secondary Cy3-coupled Ab and either an anti-albumin or anti-CK18 Ab followed by a secondary FITC-coupled Ab. Samples also were stained with an an IgG control Ab followed by a secondary FITC- and Cy3-coupled Ab.
Figure 3BM-, muscle-, or brain-derived MAPC differentiate to endothelium, neuroectoderm, and endoderm in vitro. Mouse BM-, muscle-, or brain-derived MAPC maintained as undifferentiated cells for 60, 45, and 50 PDs were induced to differentiate to endothelium, neuroectoderm, and endoderm as described in the Methods section. After 14 days, cells were fixed, stained with Abs against lineage-specific antigens, and examined by confocal microscopy. Representative example of three experiments. (A) VEGF-treated MAPC stained with an IgG control Ab, anti-vWF, or anti-CD31 Ab followed by a Cy3-coupled secondary Ab. (B) bFGF-treated MAPC stained with an anti-GFAP Ab followed by a secondary Cy3-coupled Ab plus an anti-NF-200 Ab followed by a secondary FITC-coupled Ab, or stained with an anti-Tau Ab followed by a secondary FITC-coupled Ab, or an anti-GFAP Ab followed by a secondary Cy3-coupled Ab. Samples also were stained with an IgG control Ab followed by a secondary FITC- and Cy3-coupled Ab. (C) FGF4- and HGF-treated MAPC stained with an anti-HNF-1 Ab followed by a secondary Cy3-coupled Ab and either an anti-albumin or anti-CK18 Ab followed by a secondary FITC-coupled Ab. Samples also were stained with an an IgG control Ab followed by a secondary FITC- and Cy3-coupled Ab.
Figure 3BM-, muscle-, or brain-derived MAPC differentiate to endothelium, neuroectoderm, and endoderm in vitro. Mouse BM-, muscle-, or brain-derived MAPC maintained as undifferentiated cells for 60, 45, and 50 PDs were induced to differentiate to endothelium, neuroectoderm, and endoderm as described in the Methods section. After 14 days, cells were fixed, stained with Abs against lineage-specific antigens, and examined by confocal microscopy. Representative example of three experiments. (A) VEGF-treated MAPC stained with an IgG control Ab, anti-vWF, or anti-CD31 Ab followed by a Cy3-coupled secondary Ab. (B) bFGF-treated MAPC stained with an anti-GFAP Ab followed by a secondary Cy3-coupled Ab plus an anti-NF-200 Ab followed by a secondary FITC-coupled Ab, or stained with an anti-Tau Ab followed by a secondary FITC-coupled Ab, or an anti-GFAP Ab followed by a secondary Cy3-coupled Ab. Samples also were stained with an IgG control Ab followed by a secondary FITC- and Cy3-coupled Ab. (C) FGF4- and HGF-treated MAPC stained with an anti-HNF-1 Ab followed by a secondary Cy3-coupled Ab and either an anti-albumin or anti-CK18 Ab followed by a secondary FITC-coupled Ab. Samples also were stained with an an IgG control Ab followed by a secondary FITC- and Cy3-coupled Ab.
]. After 14 days, MAPC acquired morphologic and phenotypic characteristics of astrocytes (GFAP+) and of neurons (NF-200+). The frequency of GFAP+ (approximately 25%) and NF200+ cells (approximately 40%) was similar for MAPC generated from BM, muscle, or brain. The degree of cell loss during differentiation (approximately 70%) was similar for MAPC derived from the three tissues. Finally, in neither MAPC population treated with bFGF was double labeling of cells with Abs against GFAP and NF200 seen (Fig. 3B). Cells with neuronal morphology also stained positive for Tau (Fig. 3B) and NSE (not shown).
We next tested if muscle- or brain-derived MAPC could differentiate to hepatocyte-like cells. When replated on Matrigel with FGF4 and HGF [
], 50% to 60% of muscle-, brain-, or BM-derived MAPC acquired morphologic and phenotypic characteristics of hepatocyte-like cells. Cells became epithelioid, approximately 10% of cells became binucleated, and 50% to 60% of cells stained positive for albumin, CK-18, and HNF-1 (Fig. 3C) [
Expressed gene profile of mouse BM-, muscle-, and brain-derived MAPC is highly similar
To further evaluate whether MAPC derived from different tissues were similar, we examined the expressed gene profile of BM-, muscle-, and brain-derived MAPC using U74A Affimetrix gene array. BM MAPC cultured for 45 to 120 PDs and brain and muscle MAPC cultured for 45 and 65 PDs were used. We identified genes differentially expressed in both studies. The correlation coefficient between the different MAPC populations was greater than 0.975 (Fig. 4). Comparison between the expressed gene profile in muscle MACP and BM MAPC showed that less than 1% of genes were expressed at greater than 2.2-fold different levels. Table 1A, Table 1B gives a partial list of genes that are either more highly (total number of genes = 70) or less (total number of genes = 55) expressed in muscle MAPC compared with BM MAPC. Likewise, less than 1% of genes were expressed greater than 2.2-fold different level in BM- than brain-derived MAPC. Table 1A, Table 1B also shows a partial list of genes that are either more highly (total number of genes = 98) or less (total number of genes = 99) expressed in brain MAPC compared with BM MAPC. When we evaluated all genes differentially expressed between MAPC from BM and brain, five neuroectodermal specific genes were more highly expressed in BM MAPC than brain MAPC and seven neuroectodermal specific genes were more highly expressed in brain MAPC than BM MAPC (Table 2). This was similar to what we saw comparing BM MAPC and muscle MAPC: seven neuroectodermal specific genes were more highly expressed in BM MAPC than muscle MAPC, and three neuroectodermal specific genes were more highly expressed in muscle MAPC than BM MAPC (Table 2). Significantly fewer muscle specific genes were seen to be differentially expressed. However, they again were found in either BM-, muscle-, or brain-derived MAPC (Table 2).
Figure 4Expressed gene profiles in BM-, muscle-, or brain-derived MAPC are similar. Scatter plot shows gene expression in BM, muscle, and brain MAPC. mRNA was extracted from 2 to 3 × 106 BM-, muscle-, or brain-derived MAPC cultured for 45 PDs at 0.5 to 1.5 × 103 cells/cm2. cDNA was prepared, labeled, and hybridized to the Affimetrix U74A array containing 6000 murine genes and 6000 EST clusters per the manufacturer's recommendations. Data analysis was done using GeneChip software. Less than 1% of genes from the U74A Affymetrix array were differentially expressed more than 2-fold among the three cell types.
The present study demonstrates that cells with multipotent adult progenitor characteristics can be culture-isolated from multiple different organs, namely BM, muscle, and brain. The cells have the same morphology, phenotype, and in vitro differentiation ability, and they have a highly similar expressed gene profile. MAPC were generated from tissues obtained from young animals (newborn to 3- to 4-week-old mice). Whether MAPC also can be generated from older animals has not been tested. However, we recently reported for human MAPC that the age of the donor did not influence the generation of MAPC [
Although we demonstrate here that brain and muscle contains cells capable of generating MAPC cultures, we have not yet tested whether other tissues also harbor such cells. BM, muscle, and brain are the three tissues in which cells with apparent greater plasticity than previously thought have been identified. BM contains cells that can contribute to a number of mesodermal [
]. What underlies this apparent cross-tissue differentiation is not known.
One possibility is that multiple tissue-specific stem cells coexist in different organs. This was shown for muscle, where the apparent differentiation of muscle to hematopoietic cells can be explained at least in part by the presence of CD45+ hematopoietic elements in the muscle [
]. However, there also is evidence that an even more primitive progenitor, one capable of differentiating in muscle or hematopoietic cells, may reside in muscle and contribute to hematopoiesis when placed in the correct microenvironment [
]. A second example of multiple tissue-specific stem cells residing in BM is the finding that BM contains Thy1+/β2-microglobulin−, albumin, and cytokeratin-19+ cells [
]. Whether these cells are partly responsible for the apparent plasticity of bone marrow cells in the transplantation setting is not known.
A second explanation is that, under the appropriate conditions, tissue-specific stem cells can be reprogrammed to differentiate into other tissue-specific cells, similar to what is seen in the most dramatic of reprogramming events, cloning [
]. Another possibility is that a continuum exists in tissue-specific stem cells, where the most primitive stem cell maintains characteristics akin to ES cells and differentiates in multiple tissue-specific cells under the influence of the appropriate microenvironment. Finally, there is evidence that stem cells, such as hematopoietic stem cells (HSC), circulate and reside outside the classic hematopoietic microenvironments [
]. Therefore, yet another explanation is that a single population of stem cells akin to ES cells circulates and can be found in multiple tissues.
Our finding that not only BM but also muscle and brain contain cells with multipotent characteristics could be explained by most of the mechanisms outlined. The first possibility, that all three organs contain mesodermal, neuroectodermal, and liver stem cells, is the least likely. We previously used retroviral marking to show that single MAPC derived from human and rodent BM differentiate to cells from the three germ layers. Although no retroviral marking studies were done for MAPC generated from muscle and brain, we previously showed that all MAPC progeny generated from CD45− cells derived murine BM MNC replated at 10 cells per well for greater than 50 PDs is single cell derived [
]. We speculate that MAPC generated from muscle or brain CD45− MNC subcultured at 10 cells per well also may be derived from a single cell.
Cells from the three organs may have undergone genetic reprogramming in culture, similar to what occurs in the “cloning process.” Our culture conditions are similar to those reported in two recent publications in which glial and oligodendrocyte progenitors cultured at low density without serum could be expanded without senescence in vitro and acquired functional characteristics of more primitive NSC [
]. Thus, the extended culture under low density and low serum conditions may have “reprogrammed” cells from the three organs. However, we have no evidence that transformation occurred as cytogenetic studies at monthly intervals did not show abnormalities. Because we did not select “stem cells” from the three different organs, we cannot determine whether muscle stem cells and/or NSC were “reprogrammed” to MAPC.
Of note, the cDNA arrays studies show that MAPC from the three tissues are highly homologous in expressed gene profile. It can be hypothesized that if the MAPC are the result of in vitro reprogramming, differences in expressed gene profile would be reflective of the tissue of origin. However, as shown in Table 2, no such pattern was detected.
The possibility exists that MSC, which can be found in tissues other than BM, such as subcutaneous fat [
], may be found in brain and muscle. Culture of cells from the three different tissues under conditions that favor MAPC generation from MSC may have allowed for reprogramming of MSC present in all three organs to MAPC. Studies in which phenotypically defined NSC and muscle stem cells are cultured under MAPC conditions are needed to address this possibility.
A number of characteristics of murine MAPC suggest that they may be related to ES cells. Like mouse ES cells, mouse MAPC, from BM, muscle, or brain, require LIF to be expanded [
Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site.
], like ES cells mouse BM-derived MAPC contribute to all somatic cell types when injected in a mouse blastocyst. If one accepts the hypothesis that MAPC are remnants of ES cells, then another question needs to be resolved. Do these ES-derived cells represent the most primitive of cells in all tissue-specific stem cell populations? Or is there a common pool of such stem cells, which, like HSC, reside in the BM and are mobilized in the blood and take up residence in other tissues such as muscle or brain? Phenotypic characterization of the cell present in fresh uncultured BM, and brain or muscle, is needed to address these questions.
Acknowledgements
This work was supported by NIH Grants RO1-DG67932, the Michael J. Fox Foundation, the Children's Cancer Research Fund, the Tulloch Family, and the McKnight Foundation.
References
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Muscle regeneration by bone marrow-derived myogenic progenitors.
Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site.
It was recently brought to our attention that we erroneously used the same FACS phenotype for BM cells in Figure 2 of the paper, Jiang Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, Verfaillie CM., Multipotent progenitor cells can be isolated from post-natal murine bone marrow, muscle and brain, Exp Hematol 2002;30: 896-904, 2002. It was identical to Figure 1 and Supplemental Figure 1 in the Nature paper Jiang Y, Jahagirdar B, Reinhard L, Schwartz RE, Keene CD, Ortiz X, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Largaespada DA, Verfaillie CM., Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 2002;418:41-49, published 2 months earlier, without indicating that these plots were identical either in the legend or the manuscript itself.
In the article entitled, Early patterning of the mouse embryo: Implications for hematopoietic commitment and differentiation, by Margaret H. Baron (Exp Hematol. 2005;33[9]:1015–1020) figures 1 and 2 were printed in reverse. Although the figure legends are correctly numbered, they were placed with the wrong figures. We apologize for this error.
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