Published online before print August 14, 2003, doi: 10.1161/01.RES.0000090624.04507.45
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© 2003 American Heart Association, Inc.
UltraRapid Communication |
Myoendothelial Differentiation of Human Umbilical Cord Blood–Derived Stem Cells in Ischemic Limb Tissues
From the Laboratorio di Biologia Vascolare e Terapia Genica (M.P., M.G.I., S.S., G.P.), Centro Cardiologico Monzino, IRCCS, Milan, Italy; Laboratorio di Patologia Vascolare (A.O., A.R.T., V.R., M.C.C.), Istituto Dermopatico dell’ Immacolata, IRCCS, Rome, Italy; and Cattedra di Ostetricia e Ginecologia (G.B., G.S.), Università Cattolica del Sacro Cuore, Rome, Italy.
Correspondence to Dr Maurizio Pesce, PhD, IDI-IRCCS, Via dei Monti di Creta 104, I-00167, Rome, Italy. E-mail m.pesce{at}idi.it
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Abstract |
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Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study, it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and, unexpectedly, to skeletal muscle cells in mice. In fact, the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions, CD34- cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells, thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org.
Key Words: stem cells • CD34 • trans-differentiation • angiogenesis • myogenesis
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Introduction |
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Endothelial progenitors cells (EPCs) were originally identified as a scant population of stem cells in human peripheral blood (PB), characterized by expression of CD34, vascular endothelial growth factor receptor (VEGFR2, KDR), and CD133 markers.1,2 The number of circulating EPCs in the peripheral blood is normally very low, representing less than 0.1% of the total mononuclear fraction of human PB. However, vascular trauma or ischemia have been reported to increase the number of these cells in the blood stream.2,3 In humans, cells displaying hematopoietic (HSC) stem cell activity have also been found in umbilical cord blood (UCB) where their number is higher compared with normal PB.4 UCB progenitor cells are routinely used in patients affected by major hematological disorders as an alternative to bone marrow transplantation for stem cell reconstitution.5 It has been shown that CD34+ cells obtained from human PB or UCB give rise to mature endothelial cells when cultured onto specific substrates or by stimulation with growth factors in culture.1,3 Furthermore, it has been described the ability of these cells to improve neovascularization and to increase blood flow in immunodeficient animal models of hind limb ischemia.3,4,6–8 EPCs have now become clinically relevant, as trials of therapeutic angiogenesis based on autologous bone marrow cell transplantation in patients affected by severe limb or myocardial ischemia have been recently published.9–12
In the present study, we show that UCB CD34+ cells survive and colonize tissues of Cyclosporine-A (Cs-A)–immunosuppressed mice. Analogous to previous observations using human UCB cells or bone marrow cells,1,4,13–15 we report that these cells undergo a program of endothelial differentiation and promote the formation of new blood vessels. In addition, we challenge the concept that UCB stem cells are a committed pool of hematopoietic/endothelial stem cells. In fact, we found that UCB stem cells improve muscle fibers regeneration by differentiating into myogenic cells. We extended this last observation by coculturing CD34+ cells onto mouse myoblast feeder layers. Under these conditions, CD34+ cells formed myotubes in mixed cultures, thus showing that these cells can be driven toward endothelial or myogenic differentiation pathways depending on culture conditions. Altogether, these findings suggest that UCB-derived stem cells are a multipotent stem cell population displaying wider differentiation plasticity than previously supposed and provide an indication of myogenic conversion of human EPCs.
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Materials and Methods |
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Isolation and Culture of EPCs From UCB
Placental blood was recovered in EDTA-containing bags immediately after delivery. UCB collection was performed with written approval by mothers. The age of neonates ranged between 36 and 42 weeks of gestation. Isolation of CD34+ cells was performed by MINIMACS system using the direct CD34 isolation kit (Miltenyi Biotech, catalog No. 130-046-702) according to manufacturer’s instructions, after obtaining the UCB mononuclear fraction by Ficoll-Histopaque (Sigma Immunochemicals) gradient separation. Purity of sorted cells was approximately 70% to 80% as assessed by FACS analysis using anti-CD34 and anti-CD133 antibodies labeled with phycoerythrin (PE) or fluorescein (FITC) (Figure 1C). For endothelial differentiation, MACS-sorted cells were cultured onto Laboratory-Tek chambers slides coated either with 0.1% gelatin or 20 µg/mL fibronectin (FN) in RPMI medium containing 20% FCS and processed for AC-LDL-Di uptake (10 µg/mL in culture medium for 4 hours) or immunohistochemistry after 10 days. For methylcellulose cultures, 104 CD34+ or CD34- per dish cells were plated in triplicate in methylcellulose medium (Stem Cells Technologies, catalog No. GF+H4535) containing interleukin-6 and -3 (IL-6, IL-3), stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) cytokines. After 14 days, the numbers of colonies containing granulocyte/macrophage (CFU-GM), granulocyte (CFU-G), macrophage (CFU-M), and endothelial cells (CFU-EC)7 were counted. To evaluate the expansion of stem cell–derived hematopoietic colonies, we grouped together CFU-GM, CFU-G, and CFU-M colonies as representative of the colony forming activity by clonogenic cells in both the CD34+ and CD34- MACS fractions. For coculture onto myoblasts or injection into ischemic tissues, CD34+ and CD34- cells were labeled with 1-1'-dioctadecyl-3-3-3'-3'-tertamethylindocarbocyanine-perchlorate (DiI) living dye1 at 5 µg/mL in serum-free RPMI medium for 30 minutes. After washing, cells were seeded onto a 60% confluent layer of C2C12 myoblasts for 2 days in proliferation medium (DMEM containing 20% FCS) before starving in differentiation medium (DMEM containing 2% horse serum) for 4 days. Under these conditions, before serum starvation, CD34+ cells attached over myoblasts whereas, after starvation, they fused within differentiating myotubes. In some experiments, C2C12 cells were irradiated with 10 Gy X-rays.16 Transduction of CD34+ cells and C2C12 mouse myoblast cell line with a retrovirus carrying enhanced green fluorescent protein (EGFP) was performed by incubating cells with the viral supernatant produced by Phoenix packaging cell line stable transfected with pPINCO retroviral vector,17 according to instructions described at http://www.stanford.edu/group/nolan/retroviral_systems/phx.html. Transduction efficiency ranged between 20% to 30% as evaluated by fluorescence microscope cell counting of five different fields/experiment containing freshly transduced cells.
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Expansion of CD34+ cells was performed using a serum-free medium (Stem Span, Stem cell Technologies) supplemented with 100 ng/mL Flt3-ligand, 100 ng/mL SCF, 20 ng/mL IL3, 20 ng/mL IL6 (all by Stem cell Technologies). After 7 days in culture, cells were counted and analyzed for CD34, CD133, and CD45 antigen expression by FACS analysis.
FACS Analysis
FACS analysis of MACS-sorted cells or CD34+ cells after expansion in culture was performed by incubating cells with 1 µg/mL FITC- or PE-conjugated mouse anti-human CD34 and CD133 monoclonal antibodies (BD-Pharmingen) for 20 minutes at 4°C in PBS containing 5% FCS. PE- or FITC-conjugated mouse IgGs were used as isotype control in FACS analysis at the same concentration as specific primary antibodies; 104 cells with no gate were analyzed in each sample into a FACScalibur fluorescence activated cell sorter (Beckton-Dickinson).
Animals and Surgical Procedures
Swiss CD1 male mice, 2 months old (Charles River, Italy), were used in this study. Immunosuppression was performed by injecting Cs-A at 20 mg/kg weight for 2 days before, and daily after the surgery, for the entire period of the experiment. To produce hind limb ischemia, the left femoral artery in anesthetized mice (2,2,2-tribromoethanol, 880 mmol/kg body weight, Sigma Immunochemicals) was excised with an electrocoagulator from its proximal origin as a branch of the external iliac artery till the bifurcation into saphenous and popliteal arteries as described.18 Injection of human EPCs was performed at the same time by injecting cells resuspended in 30 µL PBS at three levels (proximal, medial, distal, 10 µL each) of the adductor muscle, along the femoral artery site after its removal.
Histology
For cryosectioning, unfixed adductor muscles were removed 7 or 14 days after ischemia and immediately frozen in isopentane in liquid N2 for later inclusion in OCT compound (Bio Optica) and sectioning at 5 µm into a cryostat. For histology, mice were perfused first with 50 mL phosphate buffer (0.2 mol/L, pH 7.4) containing 5000 U/mL heparin (Roche) and then with 4% paraformaldehyde in PBS (pH 7.4) for 10 minutes. The dissected adductor muscles were embedded in paraffin and sectioned at 3 µm. Hematoxylin-eosin staining was performed in order to count muscle fibers, whereas immunohistochemistry using anti–
-actin antibody was performed to quantify arteriole length density (see next section).
Immunohistochemistry
Endothelial cells obtained in culture from CD34+ cells were analyzed by immunohistochemistry using a mouse monoclonal anti–Flk-1/KDR (clone A3, Santa Cruz) at 1 µg/mL in PBS containing 1 mg/mL BSA and 0.1% Triton X-100 (TX), followed by incubation with FITC-conjugated anti-mouse antibodies in the same medium. Factor VIII (Von Willebrand factor) immunohistochemistry was performed by incubating the cells with a rabbit polyclonal antibody (DAKO) at 10 µg/mL in TX-containing PBS-BSA followed by incubation with FITC-conjugated, anti rabbit polyclonal antibodies. Frozen section of adductor muscles from mice injected with DiI-labeled UCB CD34+ cells were first fixed by incubating with 2% paraformaldehyde in PBS for 15 minutes at room temperature and then incubated with 1 to 10 µg/mL mouse monoclonal anti-desmin (clone DE-U-10, Sigma Immunochemicals), mouse monoclonal anti–-actin (clone 1A4, Sigma Immunochemicals) or rabbit polyclonal anti laminin (Sigma Immunochemicals) antibodies in PBS containing 2% BSA. After washing, sections were stained with fluorescein-conjugated goat anti-mouse or goat anti-rabbit polyclonal antibody (DAKO) in the same medium. Human MyoD staining was performed with a specific mouse anti-human MyoD monoclonal antibody (clone G106-647, BD-Pharmingen) at 2.5 µg/mL concentration in PBS containing BSA. A biotinylated monoclonal anti-mouse antibody was used as a secondary antibody, followed by ABC complex incubation and DAB Peroxidase staining kit (Vector Laboratories). For GFP immunohistochemistry, sections were incubated with anti-GFP rabbit polyclonal antibody (AbCam, No. Ab290) at a 10 µg/mL concentration, followed by FITC-conjugated anti-rabbit antibodies. For arteriole counting, paraffin sections of adductor muscles were stained with anti-mouse smooth muscle -actin (see before) and further stained with rhodamine-conjugated goat anti-mouse polyclonal antibody (Sigma Immunochemicals). Sections were observed under a ZEISS Axiovert fluorescence microscope and images were acquired and stored with image analyzer KS300 software.
Morphometric Analysis
The method for evaluating the arteriole length density has been described previously.19,20 Briefly, sections of adductor muscles stained with anti–-actin from each mouse were examined under fluorescence microscope. The measures of major and minor diameters of each arteriole along with the arteriole wall thickness were determined and scored by KS300 imaging software. For "n" arterioles scored in a given area (A), the length density (Ld) corresponds to the sum of the ratios (Rn) between the major and the minor axes of each arteriole. Thus, Ld is equal to the length per unit volume in the same dimensional area: Ld=1/A
R, where R is (R1+R2+.....+Rn). The number of regenerating muscle fibers in the adductor muscles of saline- or cell-injected mice was calculated by counting the total number of fibers that showed centrally located nuclei.21 This number was normalized to the section area calculated with KS300 imaging software. Sections showing infiltration by T lymphocytes, likely due to rejection of human cells (Figure 2A), were not included in morphometric evaluations. All mice displaying rejection associated with necrosis of the tissues (Table) were not considered for morphometric determination of arteriole length density and muscle fiber density.
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Statistical Analysis
Cell culture experiments and FACS analyses were performed in triplicate in at least three different experiments. Significance was calculated by Student’s t test using Jandel Sigma-Stat statistical software. A P<0.05 was taken to indicate statistical significance. Results are reported as average±SE.
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Results |
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Differentiation of UCB CD34+ Cells Into Endothelial Cells In Vitro
CD34+ cell separation from the mononuclear fraction of UCB was performed using a sorting approach based on magnetic separation (see Materials and Methods). Enrichment of EPCs in the CD34+ cell fraction was evaluated by FACS analysis of CD34 and CD133 marker expression. Inset in Figure 1C shows a typical FACS profile of single CD34+ and double CD34+/CD133+ cells (total CD34+ cells: 83%). Expansion in methylcellulose medium containing cytokines was performed to confirm that the CD34+-enriched populations contained cells having the potential to differentiate into hematopoietic and endothelial cells in vitro. In these conditions, CD34+ cells gave rise to hematopoietic colonies (CFU-GM, CFU-M, and CFU-G; Figure 1A) and colonies containing endothelial cells (CFU-EC; Figure 1B),7 whereas the CD34- cells produced only a few colonies (Figure 1C). To show the ability of CD34+ cells to differentiate into endothelial cells, we performed liquid culture of enriched cells obtained by CFU-EC colonies grown in methylcellulose or freshly isolated UCB CD34+ cells onto gelatin or fibronectin-coated dishes in the presence of 20% FCS.1 In these conditions, the cells attached and spread onto the substrate and, after 7 days of culture, formed clusters of spindle-shaped cells with typical endothelial cell morphology (Figure 1D). These cells were tested for the uptake of Ac-LDL-DI substrate (Figure 1E)1 and for the expression of endothelial cells markers, ie, Von Willebrand factor (Figure 1F) and KDR receptor (Figure 1G).22,23
Injection of Human Stem Cells Into Ischemic Limbs in Immunosuppressed Mice
Previously, it has been shown in NOD/SCID immunodeficient mice1,13 or nude rats4 that EPCs from PB or UCB promote angiogenesis in vivo. To date, however, no studies of this type have been performed using immunosuppression protocols of normal animals. Therefore, 1x105 purified UCB CD34+, CD34- cells, or saline were injected into immunocompetent Swiss CD-1 mice treated with Cs-A for 2 days before, and daily after the injection of stem cells in the adductor muscle of ischemic limbs. Injection was performed at three different levels (proximal, medial, and distal) in the adductor muscle along the site of femoral artery after its removal, in a way that cells became distributed into the ischemic muscle. To evaluate rejection of injected human cells, histological sections of adductor muscles of each mouse were examined at three different levels (proximal, medial, and distal) to search for generalized inflammation of injected tissues. At 7 days after injection, only 2 mice out of 22 injected with human cells (9.1%) exhibited histological evidence of an acute inflammation (Table and Figure 2A), distinct from ischemia-induced tissue necrosis (Figure 2B) or normoperfused tissue morphology (Figure 2C). At 14 days after injection, the number of mice displaying inflammation in the adductor muscle raised to a 5:14 ratio (35.7%). This was also associated with necrosis, indicating that immunosuppression protocol failed to prevent all mice from developing a chronic rejection of human cells or graft versus host disease (GVDH).
Endothelial Differentiation Potential of UCB CD34+ Cells In Vivo
To measure the overall angiogenic effect of injected cells, we determined the length density of arterioles 4 to 41 µm in diameter24 (Figure 3A) in adductor muscles of mice injected with CD34+, CD34-, and saline, 7 and 14 days after ischemia. As shown in Figure 3B, the arteriole length density in ischemic limbs was significantly increased at both time points by CD34+ cells, whereas CD34- cells did not induce an angiogenic response compared with saline-treated mice, except for a slight increase at 14 days that, however, was significantly lower than in mice injected with CD34+ cells. To determine whether injection of stem cells contributed to angiogenesis in vivo in the absence of ischemia, UCB CD34+ cells were injected into nonischemic adductor muscles and arteriole length density was determined after 7 days. The results showed that CD34+ cells enhanced the number of arterioles also in nonischemic muscles to a level comparable to that achieved in ischemic condition, whereas CD34- cells did not have such potential.
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To allow recognition of injected human cells in mouse tissues, UCB CD34+ cells were infected with a retrovirus carrying EGFP-protein17 before being injected in the ischemic muscles.1 Transduction efficiency of these cells ranged between 20% and 30%, as evaluated by fluorescence microscope examination of living cells (not shown, see Materials and Methods). Thereafter, injected cells in recipient tissues were identified by immunohistochemistry for GFP in fluorescence microscopy analysis of muscle cross sections. We found that a number of arterioles contained GFP+ cells (Figure 3C). To quantitatively measure the contribution of UCB CD34+ cells to endothelial cells (ECs) formation in stem cell injected mice, GFP+ endothelial cells were counted in adductor muscle sections triple stained with -actin antibodies, GFP antibodies and Hoechst nuclear dye. We calculated the average number of GFP+ EC cells compared with the average number of total EC cells in the same arterioles. The results of cell counting performed in a total of 97 arterioles (range 11 to 41 µm) in 3 different mice injected with GFP+ CD34+ cells revealed an average of 1.66±0.21 GFP+ ECs/arteriole in a total of 5.85±1.22 ECs/arteriole (29%). It has been recently reported that peripheral blood contains precursors of vascular smooth muscle cells (VSMCs).25,26 To investigate whether injected UCB cells differentiated into VSMCs, we screened for double GFP+/-actin+ cells in the arteriole wall (Figure 3E). GFP+/-actin+ cells were sporadically noticed into the arteriole wall suggesting that, although possible, CD34+ cells were not preferentially converted into VSMCs.
Angiogenic Potential of Ex Vivo–Expanded CD34+ Cells
A possible limitation to the use of UCB or PB-derived EPCs for therapeutic angiogenesis, is the relatively low number of CD34+ cells that can be recovered from fresh cord blood samples. To overcome this problem, expansion in culture of CD34+-derived endothelial cells has been proposed as a useful strategy to increase the amount of differentiated endothelial cells for blood vessels regeneration.6 An alternative to this method that was not assessed before is expansion of EPCs in conditions that should preserve their differentiation potency. In fact, several studies have described the use of cocktails of cytokines allowing rapid expansion of HSCs in culture and preserving their engraftment ability in the bone marrow of recipients. Accordingly, UCB CD34+ cells were grown in serum-free medium containing SCF, Flt3 ligand, IL-3, and IL-6 cytokines (adapted from Lazzari et al27). In this medium, cell number increased about 10 times in 7 days of culture (not shown) and expression of CD34 antigen was maintained (50.5%±3.16% CD34+ cells after expansion for 7 days). Ex vivo expanded cells maintained an in vivo angiogenic activity to a level comparable to freshly isolated cells at 7 days after ischemia (Figure 3).
Injection of UCB CD34+ Cells Accelerates Muscle Repair In Vivo
Microscopic examination of mouse adductor muscles injected with DiI-labeled CD34+ cells revealed that a number of such cells were not included in newly formed capillaries or arterioles, but were likely localized within muscle fibers (Figure 4A), suggesting that they may be recruited in muscle cell compartment and contribute to muscle repair in ischemic limbs. The results of an immunofluorescence assay on frozen sections of adductor muscles injected with DiI-stained CD34+ cells revealed that some cells expressed desmin (Figures 4B through 4D), a marker for activated satellite cells.28
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Limb ischemia is a condition characterized by degeneration of vascular and skeletal muscle tissues due to blood flow deprivation (Figure 2B). Hypoxia is one of the major causes of cell death. Regeneration of muscle fibers after ischemia parallels restoration of blood flow by formation of new blood vessels.29 To assess whether injection of CD34+ cells enhanced muscle regeneration, we determined the density of regenerating muscle fibers in the adductor muscles at 7 days after ischemia. Regenerating muscle fibers can be easily recognized morphologically, due to the presence of nuclei located in the center of the fibers compared with fully developed fibers where the nuclei are located close to the sarcolemma21,30 (compare Figures 5A and 5B). In tissues treated with freshly isolated CD34+ cells, the density of regenerating fibers was markedly higher compared with controls. Analogous to endothelial differentiation, CD34- did not promote myogenic repair, whereas in vitro expanded EPCs improved myogenesis (Figure 5C). Myogenic regeneration in nonischemic muscles injected with either CD34+ or CD34- cells was also analyzed. Interestingly, as shown in Figure 5C, injection of CD34+ or CD34- cells into normoperfused muscles did not lead to formation of new muscle fibers. The analysis of muscle regeneration was not extended beyond day 7 because spontaneous regeneration of adductor muscles in ischemic limbs typically peaks between day 14 and 21 after ischemia (not shown), making it impossible to detect a further myogenic effect of CD34+ cells injection.
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Direct Participation of CD34+ Cells to Muscle Cell Generation In Vivo and In Vitro
Studies using MyoD-LacZ transgenic mice have shown expression of MyoD in the nuclei of activated muscle satellite cells and the newly formed muscle fibers.21 Therefore, detection of human MyoD (hMyoD) expression in the nuclei of UCB CD34+-derived cells should allow to test whether injected cells undergo a program of myogenic differentiation. The expression of hMyoD transcription factor in regenerating fibers of mice injected with either CD34+ or CD34- cells was then studied by immunohistochemistry. For these experiments, we used an antibody that specifically recognizes human but not mouse MyoD (see Materials and Methods). hMyoD staining was found in the nuclei of regenerating muscle fibers (Figure 6A) and in cells that are juxtaposed to the muscle fibers in mice injected with CD34+ cells (Figure 6B). To visualize the position of these cells within the muscle fibers, we performed immunofluorescence on consecutive sections using anti-hMyoD and anti-laminin antibodies (Figures 6F and 6G). By this analysis, we observed that the hMyoD+ cells were localized outside the basal lamina. Interestingly, some regenerating myofibers contained stained and unstained nuclei (Figure 6C), suggesting that CD34+ cells formed mixed human/mouse fibers. Mice injected with CD34- cells did not show hMyoD staining in regenerating muscle fibers (not shown). To further assess UCB CD34+ cell direct contribution to muscle regeneration, we analyzed sections of adductor muscles of mice injected with GFP-labeled CD34+ cells. As shown in Figure 6E, we found small GFP+ regenerating fibers. These muscle fibers were quantified by counting the number of GFP+ fibers in the total number of regenerating fibers in each section. In a total of 4072 regenerating muscle fibers counted in different sections from 4 mice injected with GFP+ CD34+ cells, we found 232 to be GFP+ (5.6%; Figure 6E). Altogether, these results suggest that UCB CD34+ cells have the ability to directly participate to myogenesis in vivo.
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In vitro experiments were performed in order to assess the potential of UCB CD34+ cells to differentiate into myogenic cells. According to this hypothesis, DiI-labeled CD34+ and CD34- cells were seeded onto a layer preformed with a line of mouse myoblasts, the C2C12 cells, stable transduced with a retrovirus driving GFP expression. This cell line has been previously reported to sustain myogenic differentiation of muscle-derived side-population (SP) cells.31 In the presence of 20% FCS (proliferation medium), C2C12 cells proliferate, whereas after starving in 2% horse serum (differentiation medium), C2C12 cells become postmitotic and fuse to form myotubes.32 During 2 days of culture after seeding, CD34+ cells attached to proliferating myoblasts. Due to coculture condition, it was not possible to determine by quantitative assays whether UCB CD34+ cells proliferated over the layer of cycling myoblasts. However, switching the cultures to differentiation medium caused the appearance of several DiI+/GFP+ myotubes, suggesting myogenic differentiation of UCB CD34+ cells in vitro (Figures 7A and 7B). Because the formation of polynucleate myotubes in culture is a physiological consequence of cell fusion, it is likely that diffusion of both DiI and GFP occurred within myotubes formed by a mixture of DiI-labeled (CD34+) and GFP-labeled (C2C12) cells (Figures 7C through 7E). A quantification of in vitro myogenic activity was performed by counting the number of DiI+ myotubes obtained by seeding an equal number of CD34+ and CD34- cells onto GFP-labeled myoblasts. As shown in Figure 7F, the number of DiI+ myotubes obtained with CD34+ cells was significantly higher than with CD34- cells, showing that myogenic activity is mostly confined in the CD34+ cell population of human UCB. Recently, it has been found that fusion of neuronal and hematopoietic stem cells to ES cells probably accounts for dedifferentiation.33,34 For this reason, we assessed the contribution of spontaneous fusion in coculture of DiI+ CD34+ cells onto GFP+ myoblasts before serum starvation. To prevent possible interference by spontaneous myogenic differentiation of C2C12 cells in proliferation medium, we treated feeder cells with 10 Gy ionizing radiation before seeding CD34+ cells. This treatment has been shown to interfere with the normal program of myoblast myogenic differentiation,16 thereby allowing discrimination between myoblast and UCB stem cell fusion. In this assay, we found a majority of cells that were stained only with DiI and a significantly lower number of DiI+/GFP+ cells (Figures 8A through 8C). We further quantified CD34+ cell-induced fusion by counting the number of DiI+/GFP+ versus DiI+ only cells (Figure 8D). This showed that the number of DiI+ cells was significantly higher than double DiI/GFP-labeled cells, thus suggesting a relatively low rate of CD34+ cell fusion to C2C12 cells. Unfortunately, due to secondary death of irradiated C2C12 cells, it was not possible to determine whether UCB cells seeded onto irradiated C2C12 cells formed myotubes in differentiation medium. In additional experiments, we thus assessed whether CD34+ cells form myotubes when cocultured onto normal GFP+ myoblasts by counting the number of DiI+ myotubes (Figure 8E) and DiI+/GFP+ (Figure 8F) myotubes after serum starvation (Figure 8G). Both CD34+ and CD34- cells gave rise to a number of double-positive myotubes. However, the number of DiI+ only myotubes obtained by seeding CD34+ was higher than with CD34- cells. This result, in line with the evidence of a higher myogenic activity in vivo (Figure 5) and in vitro (Figure 7), again suggests that UCB CD34+ cells are capable of myogenic commitment independent of cell fusion.
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Discussion |
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EPCs in the peripheral blood or the umbilical cord blood are a potential source of stem cells from humans to enhance angiogenesis in ischemic tissues. Recent clinical trials of autologous EPC transplantation in patients affected by heart and limb ischemia have recently shown the potential therapeutic benefits of stem cell transplantation to enhance angiogenesis.9–12 Except for these trials in humans, the potential of hEPCs to generate blood vessels in vivo has been assessed so far in immunodeficient mouse1,3,6,13 and rat hind limb4 or heart8 ischemia models.
UCB CD34+ Cell Injection in Cs-A–Treated Mice Does Not Cause Massive Rejection but Enhances Angiogenesis
The assessment of transplanted human cell rejection was performed by analyzing histological sections of adductor muscles in the proximity of cell injection sites. Except for 2 of 22 cases at day 7 (documented in Figure 2 and the Table), lymphocyte infiltration, necrosis, and tissue fibrosis were comparable to saline-treated mice after ischemia. A relative increase in cell graft failure was noticed at day 14, when about 36% of mice receiving human cells showed signs of generalized tissue inflammation (Table). This suggests that treatment with Cs-A was not completely effective in preventing a certain degree of rejection of human cells or development of GVHD. Because the evaluation of arteriole and muscle fibers density may be altered by tissue inflammation, mice displaying tissue inflammation were excluded from morphometric analyses of blood vessel and/or muscle regeneration (Table). Acute rejection is normally due to the presence of anti-species antibodies in the host. These antibodies mediate organ xenograft failure due to complement activation on endothelial cells of transplanted organs followed by thrombosis. This represents to date one of the major hurdles to the use of interspecies organ transplantation. There are two possible explanations for our finding that only moderate rejection occurred in the ischemic muscles of mice injected with human CD34+ cells. First, transplanted EPCs are not mature endothelial cells and may represent suboptimal targets for the action of preformed anti-species antibody. Second, regenerating blood vessels and muscle fibers likely consisted of mixed host/donor structures (Figures 3 and 6
). This may, in part, justify a lower immunogenic potential of neoformed tissues and longer tolerance by the hosts. A discrepancy between our results and previous observations has been found in the different angiogenic response to CD34+ cell injection in nonischemic mice. In fact, it was described that endothelial commitment of EPCs is specifically linked to ischemia4 or tissue damage.35 Besides being recruited by angiogenic factors in ischemic tissues, and taking part to angiogenesis by differentiating into endothelial cells, EPCs per se promote angiogenesis by releasing proangiogenic molecules such as VEGF15 and angiopoietin136 that act on preexisting angioblasts or endothelial cells. One possible explanation for the discrepancies between the present and prior observations4 is that in the strain of mice used in the present study, responsiveness of host angioblasts and/or endothelial cells to stem cell–produced cytokines is higher than in immunodeficient SCID or inbred mice used previously. Our finding that in a model of immunosuppression in mice the ability of CD34+ cells to differentiate into endothelial cells and to promote the formation of blood vessels in ischemic limbs is maintained represents an advancement toward validation of use of EPCs for allotransplantation in patients.
Myoendothelial Differentiation Pathways of CD34+ Cells: Environmental Control of a Multipotent Cell Type Reprogramming?
Several investigations have shown the existence of different stem cell population in the muscle and the bone marrow that share common markers and may be thus lineage-related. As an example, in mice and humans, CD34 is expressed in circulating and UCB endothelial precursor cells,1,4,8 but it is also known as a quiescent satellite cell marker in the skeletal muscle and it is expressed in these cells together with myogenic master gene Myf-5.37 Both skeletal muscle and bone marrow contain a population of cells characterized by low staining by Hoechst 33342 and rhodamine 123 that are thereby called "side population" (SP).38 Stem cell activity of SP muscle stem cells (MSCs) is not restricted to the myogenic phenotype, as SP cells from the muscle participate to bone marrow reconstitution in lethally irradiated mice.39 Conversely, bone marrow stem cells are able to differentiate into myogenic cells39,40 and take part to both cardiac and skeletal muscle regeneration after injury.41–43 A recent study has described in mice a myoendothelial precursor cell type expressing CD34 in the interstitial space between muscle fibers.44 These cells have both myogenic and endothelial cell differentiation potency in culture, but in their undifferentiated state, they only express CD34 and Brcp1 gene product, which is one of the proteins involved in the acquisition of the "side population" phenotype of HSCs.45 Notably, these cells do not express myogenic markers, but upregulate myogenic master genes such as Pax3 and Pax7 and give rise to mixed myoblast/endothelial colonies in clonal cultures. It is likely that these progenitors represent a myoendothelial stem cell type deriving from either a pool of circulating HSCs or from progenitor cells deriving from cells migrating in the muscle from the dorsal aorta during embryogenesis.40,44,46 Our results show that an increase in myogenic regeneration by UCB CD34+ cells injection paralleled the increased formation of new arterioles in ischemic tissue. Thus, it is possible that the increase in muscle regeneration was only the consequence of angioblast-mediated recovery of blood flow in the ischemic tissue. As shown in Figures 4 and 6 C, however, the presence of desmin+ cells between muscle fibers or the simultaneous presence of nuclei stained with anti-human MyoD protein in regenerating muscle fibers or the occurrence of GFP+ muscle fibers in mice injected with GFP-labeled CD34+ cells suggests that, at least in part, and similar to a recent mouse model of bone marrow progenitor cell recruitment in the muscle stem cell compartment,40 CD34+ cells participate to myogenic regeneration after ischemia. In particular, the presence of hMyoD+ cells localized outside the fiber basal lamina (Figure 6B, 6F, and 6G), suggests that a pool of CD34+-derived cells may differentiate into myogenic cells before being recruited into satellite cells pool. These cells, consistently with the observations raised by Tamaki et al47 in rapidly growing muscles, may be MyoD+ cells derived from injected CD34+ cells that are involved in the formation of new fibers in the interstitial space.
The quantitative evaluation of GFP+-regenerating muscle fibers (Figure 6) showed that only about 6% of such fibers showed a contribution by injected human cells. This may be explained by the relatively low efficiency in retroviral transduction of CD34+ cells, but also by an underestimation of the effective contribution of CD34+ cells to heal ischemic muscles, due to histological examination of syncytial muscle fibers in transverse sections. In addition, it is possible that only a subset of CD34+ cells is capable of myogenic conversion. Altogether, the results of the present study support the hypothesis that, analogous to the muscle regenerating activity of mesenchymal stem cells48 or muscle derived stem cells in dystrophic mice (for a review, see Jankowski et al49), muscle regeneration after ischemia is induced through a certain degree of direct differentiation of UCB CD34+ cell into myogenic cells.
Trans-Differentiation Rather Than Fusion Accounts for Myogenic Commitment of CD34+ Cells
Figure 7 suggests that CD34+ cells cocultured with mouse myoblasts give rise to muscle cells. This is surprising, as recent work has convincingly shown that mouse bone marrow SP cells cannot be reprogrammed into myogenic cells by coculture onto muscle cells in vitro.31 One possibility is that UCB angioblasts have a different extent of plasticity compared to mouse bone marrow cells. In fact, it has been reported that human EPCs derived from peripheral blood CD34+ cells50 and that UCB CD34+ cell fraction contains a higher number of primitive hematopoietic cells compared with human bone marrow.51 In our analysis, we attempted at deriving human myogenic cells by using conditioned media by both proliferating and differentiating myoblasts (not shown). Thus far, in agreement with results described by Asakura et al,31 we could not derive any myogenic cells either by using liquid culture or semisolid culture in methylcellulose even in the presence of myoblast conditioned medium. This suggests that cell contact is probably necessary to induce myogenic phenotype in blood borne stem cells. We are currently unaware of whether, analogous to cardiac myocyte conversion of human EPCs50 or endothelial cells,52 cell contact is also sufficient to induce myogenic conversion of human EPCs. Work is now in progress to dissect this mechanism by using combinations of adhesion molecules, diffusible factors, and genetic modification of stem cells by viral gene transfer.
Recent results obtained by coculturing somatic stem cells with embryonic stem cells has raised the important issue that spontaneous fusion may account for somatic stem cell dedifferentiation or reprogramming.33,34 Myogenic differentiation of hematopoietic stem cells, in particular, is potentially subject to interference by fusion since terminal differentiation of muscle fibers in vivo and myotubes in vitro implicates formation of syncytial myotubes as a result of fusion between donor-derived stem cells and myogenic cells in the host. Thus, it may be inferred that formation of regenerating fibers containing donor-derived nuclei stained for a myogenic marker such as MyoD or even appearance of GFP+ fibers (Figure 6) may result from fusion rather than to a real reprogramming event. On the other hand, we surmise that spontaneous fusion does not entirely account for the process of CD34+ cell-mediated muscle repair as we found that (1) increase of myogenesis only occurred by injecting ischemic mice with CD34+ cells (Figure 5), (2) formation of myotubes in culture was higher by culturing CD34+ cells onto myoblasts compared with CD34- cells (Figure 7F), (3) the majority of CD34+-derived cells in coculture were not fused to myoblasts before induction of myogenic differentiation by serum deprivation (Figures 8A through 8D), and (4) DiI+ only myotubes were more frequently scored by seeding CD34+, rather than CD34- cells, onto GFP-labeled C2C12 myoblasts (Figure 8G). In addition, generation of new muscle fibers only occurred in tissues damaged by ischemia (Figure 5). Taken together these findings suggest that determination of myogenic phenotype is specific for CD34+ EPCs and that fusion with preexisting myogenic cells in vivo (Figure 6) occurs preferentially under stimulation by physiological signals that are upregulated on tissue damage, or in vitro (Figure 7) following a differentiative trigger such as serum deprivation.
Conclusion
The existence of peripheral blood/bone marrow stem cell plasticity is currently hotly debated. Numerous studies have provided both positive and negative demonstrations of bone marrow stem cell conversion into skeletal and cardiac myocytes, and its potential clinical relevance.31,35,39,41,53,54 It has been hypothesized that tissue damage is one of the conditions that might be involved in stem cell lineage barrier transition and trans-differentiation.55 Our findings, showing that human CD34+ cells may induce angiogenesis also in nonischemic mice, suggest that an angioblast "default" differentiation pathway into endothelial cells is activated even without damage-associated stimuli. By contrast, induction of myogenic phenotype in CD34+ cells appears to be exclusively linked to the presence of ischemic condition in host tissue. This observation is in line with evidences showing that contribution of recruited hematopoietic stem cells to skeletal and cardiac muscle repair is strongly enhanced by tissue injury.40,56 At present, the identity of factors that commit CD34+ stem cells toward endothelial and/or myogenic phenotype in ischemic tissues is controversial. However, an array of cytokines and chemokines (VEGF, PlGF, SDF-1, GM-CSF, SCF, and HGF) that are involved in mobilization of EPCs in the peripheral blood after ischemia or after plasmid or viral vector-mediated gene transfer have been identified.57–60 It is still a matter of speculation whether multiple signaling by these factors or an interplay between these factors and intracellular pathways activated by adhesion molecules such as integrins, inflammatory cytokines, or inducing signals, eg, Wnt signaling, play a role in ischemia-induced recruitment and, possibly, trans-differentiation of stem cells. Future studies addressing this question are therefore necessary to unravel how stem cell–mediated regeneration may be triggered to heal ischemic tissues.
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Acknowledgments |
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This work was supported by (1) Italian Ministry of Health, Ricerca Finalizzata: "Proliferazione e transdifferenziamento di cellule staminali in terapia cellulare," contract No. 186 and (2) Ricerca Corrente, Centro Cardiologico Monzino, research program: "Biochimica Clinica Sperimentale," project No. 7.4: "Nuovi approcci di terapia cellulare della patologia ischemica degli arti inferiori."
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Footnotes |
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*Both authors contributed equally to this work.
Original received June 18, 2003; resubmission received July 24, 2003; accepted July 29, 2003.
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