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Low angiogenic potency induced by the implantation of ex vivo expanded CD117⁺ stem cells

Department of Medical Bioregulation, Division of Cardiovascular Surgery and Medicine, Yamaguchi University School of Medicine, Yamaguchi 755-8505, Japan

Submitted 9 October 2003 ; accepted in final form 24 November 2003

	ABSTRACT

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Ex vivo expansion of stem cells might be a feasible method of resolving the problem of limited cell supply in cell-based therapy. The implantation of expanded CD34⁺ endothelial progenitor cells has the capacity to induce angiogenesis. In this study, we tried to induce angiogenesis by implanting expanded CD117⁺ stem cells derived from mouse bone marrow. After 2 wk of culture with the addition of several growth factors, the CD117⁺ stem cells expanded 20-fold and had an endothelial phenotype with high expression of CD34 and vascular endothelial-cadherin. However, >70% of these ex vivo expanded cells had a senescent phenotype by -galactosidase staining, and their survival and incorporation were poor after implantation into the ischemic limbs of mice. Compared with the PBS injection only, the microvessel density and the percentage of limb blood flow were significantly higher after the implantation of 2 x 10⁵ freshly collected CD117⁺ cells (P < 0.01) but not after the implantation of 2 x 10⁵ expanded CD117⁺ cells (P > 0.05). These data indicate that ex vivo expansion of CD117⁺ stem cells has low potency for inducing therapeutic angiogenesis, which might be related to the cellular senescence during ex vivo expansion.

ischemic; blood flow; senescence; differentiation

Several stem cell sources, derived from peripheral blood, bone marrow, or embryonic stem cells, have been used to induce angiogenesis experimentally (1, 7, 11, 12, 14, 16, 17, 21). Stem cells from peripheral blood or bone marrow are one of the most viable cell populations for clinical application because the implantation of autologous cells is not associated with problems of immunological rejection or ethical conflict. Some clinical trials have been done on implanting autologous CD34⁺ peripheral blood stem cells or bone marrow mononuclear cells to induce therapeutic angiogenesis for the treatment of ischemic heart or limb diseases (2, 5, 6, 8, 22, 25–28). The major problem associated with using autologous stem cells is limited supply because stem cells are a rare in peripheral blood and bone marrow.

Ex vivo expansion could resolve this problem. It was reported (13, 15) that the implantation of ex vivo expanded CD34⁺ cells has the potential to induce therapeutic angiogenesis. Because there are more stem cells in bone marrow than in peripheral blood (3), we decided to separate stem cells from bone marrow for ex vivo expansion. Furthermore, we studied CD117⁺ cells rather than CD34⁺ cells because some of the CD34^– stem cells in bone marrow exhibit potential for proliferation and endothelial differentiation (10, 23), and the CD117⁺ cells in bone marrow play a key role in inducing angiogenesis (19).

In this study, we expanded CD117⁺ stem cells by culture supplemented with several cytokines and then injected these ex vivo expanded cells intramuscularly into the ischemic hindlimbs of mice. Unexpectedly, compared with freshly isolated CD117⁺ cells, these expanded CD117⁺ cells showed a very low potential for inducing therapeutic angiogenesis, which might be related with cellular senescence during ex vivo expansion.

	METHODS

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Animals. Male 12- to 15-wk-old C57BL/6 mice were used for these experiments, which were approved by the Institutional Animal Care and Use Committee of Yamaguchi University. The animals were bred in clean conditions and allowed free access to food and water in a temperature-controlled environment with a 12:12-h light-dark cycle.

Separation and culture of CD117⁺ cells. Bone marrow cells were collected from the femur and tibia, and mononuclear cells were isolated by density gradient centrifugation. CD117⁺ cells were separated using a magnetic cell sorting system, as described previously (19). Approximately 2.5% (1.6% to 3.2%) of the bone marrow cells expressed CD117, and the purity was 90% by flow cytometry determination. These isolated CD117⁺ cells have expressed 99% of CD45, 30% of CD34, and <0.5% of vascular endothelial (VE)-cadherin.

Isolated CD117⁺ cells were plated on culture dishes coated with 1% gelatin (Sigma) at a density of 2 x 10⁵ cells/ml in RPMI 1640 medium supplemented with 15% fetal bovine serum (GIBCO), 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO). Cells were incubated at 37°C in 5% CO₂. The following cytokines were added to the media: 50 ng/ml vascular endothelial growth factor, 5 ng/ml fibroblast growth factor, 5 ng/ml insulin-like growth factor-1, and 10 ng/ml stem cell factor. Additional feeding was performed every 3 days. After 3, 7, 14, 21, and 28 days of culture, CD117-derived adherent cells were harvested and the total number of surviving cells was counted. Because the proliferation of CD117⁺ cells became poor after 14 days of culture, we collected expanded CD117⁺ cells for the following study 14 days after cultivation.

For immunostaining analysis of endothelial differentiation, cells were seeded on 4-well chamber culture slides (Nalge Nunc International) coated with 1% gelatin and cultured as described above. After 14 days of culture, the cells were fixed in 1% formaldehyde and blocked with 2% BSA and then incubated with FITC-conjugated antibodies against CD34 (Pharmingen).

Flow cytometry. To characterize these ex vivo expanded CD117⁺ cells, cells (10⁵) were harvested after 14 days of culture and then incubated for 30 min at 4°C with the following antibodies: FITC-conjugated monoclonal antibodies against CD34, VE-cadherin, and fetal liver kinase (Flk)-1 (Pharmingen), the FITC-conjugated CD3, CD14, and CD68 (Becton Dickinson). Isotype-identical antibodies served as controls. Quantitative flow cytometric analysis was done with the use of a FACScan flow cytometer and Cell Quest software (Becton Dickinson).

Senescence-associated -galactosidase staining. After 7 and 14 days of culture, senescence-associated -galactosidase staining was carried out as described previously (4). The number of senescence-associated (SA) -galactosidase-positive cells was counted randomly under a microscope using x200-fold magnification by a single observer and expressed as a percentage of all cells counted.

Ischemic hindlimb model and cell transplantation. The mouse ischemic hindlimb model was created as described previously (19). Briefly, after the mice were given general anesthesia, the left femoral artery was exposed and ligated, and its branches were dissected free and excised. Thirty-six mice were divided randomly into three groups, and the quadriceps and adductor muscles of the ischemic hindlimb were injected at four points with: 2 x 10⁵ freshly isolated CD117⁺ cells (Fresh group); 2 x 10⁵ expanded CD117⁺ cells (Expanded group); or PBS injection only (PBS group). The induction of angiogenesis was estimated by histological analysis and blood flow assessment 2 wk after treatment.

To examine survival, endothelial differentiation, and the incorporation of cells after implantation, cells were labeled with intracellular fluorescent dye of 5(6)-carboxyfluorescein diacetate succinimidylester (CFSE) (Molecular Plobes) as described previously (19) and then injected into the ischemic hindlimbs of 20 supplementary mice divided into two groups as described above, excluding the PBS group.

Histological assessment. Histological analysis of microvessel density was done 2 wk after treatment, as described previously (19). Briefly, five mice from each group were euthanized and the quadriceps and adductor muscles were harvested. Frozen sections of 5 µm thickness were stained for alkaline phosphatase with an indoxyl tetrazolium method and then counterstained with eosin. The number of microvessels and muscle fibers were counted under a microscope using x200-fold magnification by a single observer blind to the treatment regimen, and a total of 20 different fields on two independent slides from different cross sections were randomly selected for each mouse. The density of microvessels was estimated by the microvessel/muscle fiber ratio.

Five mice from each group were killed 7 and 14 days after the implantation of CFSE-labeled cells. Frozen sections were used to detect the survival of cells, and the microvessels were stained with R-phycoerythrin-conjugated anti-mouse CD34 antibody (Pharmingen) to examine cell survival, endothelial differentiation, and incorporation from the implanted cells.

Measurement of blood flow in the ischemic hindlimbs. Blood flow of the ischemic hindlimb was measured using the method of microsphere assessment as described previously (19). Seven mice from each group were reanesthetized 2 wk after treatment, and 6 x 10⁴/20 µl of eosin Dye-Trak microspheres (15 µm in diameter, Triton Technology) were injected into the abdominal aorta. The mice were euthanized 30 s later by severance of the abdominal aorta. Tissue specimens were collected from the hindlimbs and weighed and then digested in 2 M KOH containing 0.5% Tween 80, for 24 h at 60°C. The microspheres in the tissues were reclaimed, and dye from the microspheres was extracted with dimethyl formamide. The optical density (OD) of these dye samples was measured with a spectrophotometer. The recovery of perfusion in the ischemic hindlimb was estimated by comparing the percentage of limb blood flow (%LBF) with that in the normal right hindlimb, which was calculated as (OD of the ischemic limb/OD of the normal limb) x (tissue weight of the normal limb/tissue weight of the ischemic limb) x 100 (19).

Data analysis. All data are presented as means ± SD. Statistical significance was evaluated by ANOVA, followed by Scheffé's procedure and by repeated ANOVA to test for interactions. P < 0.05 was considered significant.

	RESULTS

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Characterization of ex vivo expanded CD117⁺ cells. Many CD117⁺ cells attached to the culture flask and showed a spindle-shaped morphology after 14 days of culture (Fig. 1A). These cells also showed high expression of CD34 by immunostaining analysis (Fig. 1B). Quantitative analysis showed that the CD117⁺ cells expanded by 20-fold within 2 wk of cultivation but that they had low proliferative potential thereafter, although several growth factors were supplied continuously (Fig. 1C). Flow cytometry analysis also showed that these expanded CD117⁺ cells have the qualitative properties of endothelial cells, with 70% expression of several endothelial cell-specific antigens, including CD34 (70.2% ± 6.9%), VE-cadherin (66.7% ± 7.5%), and Flk-1 (62.4% ± 4.6%). Positive expression of CD3 (1.2% ± 0.5%), CD14 (3.9% ± 1.1%), and CD68 (1.8% ± 0.6%) was seen in <5% of the cells (Fig. 2). These results indicated that endothelial differentiation was induced during the culture of CD117⁺ cells.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Ex vivo expansion of CD117+ cells. A: these CD117+ cells attached to the culture flask and showed a spindle-shaped, endothelial cell (EC)-like morphology after 14 days of culture. B: immunostaining analysis showed that almost 80% of the cells were CD34+ (data are representative of five independent experiments). C: quantitative analysis showed that the CD117+ cells expanded by 20-fold within 2 wk of cultivation but had poor proliferative potential thereafter.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Characterization of ex vivo expanded CD117+ cells. After 14 days of culture, cells were stained with FITC-labeled antibodies against CD34, vascular endothelial (VE)-cadherin, fetal liver kinase (Flk)-1, CD3, CD14, and CD68 (thick lines), or with control antibodies (thin lines). More than 70% of the cells expressed the endothelial-specific antigens CD34, VE-cadherin, and Flk-1, but they were not significantly contaminated by hematopoietic lineage cells (data are representative of five independent experiments).

Senescence of expanded cells. Senescence-associated -galactosidase staining revealed SA -galactosidase positivity in 10% of the cells (11.4 ± 4.8%) after 7 days of culture (Fig. 3A) but in >70% (73.2 ± 10.7%) after 14 days of culture (Fig. 3B). It was clearly evident that the senescence phenotype of expanded cells increased with the time of culture.

View larger version (72K): [in this window] [in a new window]   Fig. 3. Senescence of ex vivo expanded CD117+ cells. About 12% of the cells showed positive staining for senescence-associated -galactosidase activity after 7 days of culture (A), but >70% of the cells showed positive staining for senescence-associated -galactosidase activity after 14 days of culture (B) (x200 magnification).

Survival and endothelial differentiation of cells after implantation. We examined survival of the CFSE-labeled CD117-derived cells in tissue sections from the ischemic hindlimbs. Microvessels were seen by immunostaining with R-phycoerythrin-conjugated antibody against mouse CD34, and colocalization confirmed the incorporation of implanted cells into microvessels. We found that many of the cells survived, showed endothelial differentiated, and were incorporated in microvessels 14 days after implantation, in the Fresh group (Fig. 4, A–C). However, lower cell survival and incorporation were seen in the Expanded group (Fig. 4, D–F).

View larger version (13K): [in this window] [in a new window]   Fig. 4. Survival and the incorporation of cells 14 days after implantation. Representative photomicrographs show the survival of 5(6)-carboxyfluorecein diacetate succinimidyl-ester (CFSE)-labeled cells (A, D) and microvessel staining with the anti-CD34 antibody (B, E) in the ischemic hindlimbs. Numerous cells survived and were incorporated into the foci of neovascularization in the Fresh groups (C), but fewer cells were seen in the Expanded group (F).

Microvessel density in ischemic hindlimbs. After 2 wk of treatment, the microvessel/muscle fiber ratio, an index of neovascularization, was significantly higher in the Fresh group than in the PBS group (1.18 ± 0.11 vs. 0.89 ± 0.08, P < 0.01), but there was no significant difference between the Expanded and PBS groups (0.94 ± 0.12 vs. 0.89 ± 0.08, P > 0.05; Fig. 5).

View larger version (60K): [in this window] [in a new window]   Fig. 5. Histological evaluation of neovascularization in the ischemic hindlimbs 2 wk after treatment. More microvessels were seen the Fresh (B) groups than in the PBS group (A) and Expanded group (C). Compared with the mice receiving PBS injection only, quantitative analysis (D) showed that the capillary density was significantly increased in the Fresh group but not in the Expanded group (*P < 0.01 vs. PBS group).

Blood flow in ischemic hindlimbs after treatment. Blood flow of the ischemic hindlimb was estimated 2 wk after treatment. The LBF% was significantly higher in the Fresh group than in the PBS group (81.0 ± 9.8% vs. 57.5 ± 9.7%, P < 0.003) but not in the Expanded group (60.0 ± 10.3% vs. 57.5 ± 9.7%, P > 0.05; Fig. 6).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Blood flow of an ischemic hindlimb 2 wk after treatment. Compared with the mice receiving PBS injection only, the percent limb blood flow (%LBF) was significantly better in the Fresh group, but it was not significantly increased in the Expanded group (*P < 0.01 vs. PBS group).

	DISCUSSION

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This study examined the capacity of ex vivo expanded bone marrow-derived CD117⁺ stem cells to induce therapeutic angiogenesis. With the addition of several growth factors, these isolated CD117⁺ cells expanded well, by >20-fold within 14 days, and showed high expression of several endothelial markers, indicating endothelial differentiation. Furthermore, the lack of expression of CD3, CD14, and CD68 cells indicated that there was no contamination of lymphocytes and macrophages although CD117⁺ cells were well known generally as the population of hematopoietic stem cells. Although we achieved only 20-fold expansion of CD117⁺ cells, these ex vivo expanded cells showed similar characteristics and phenotypes to the expanded CD34⁺ cells reported by Kalka et al. (13). These findings suggest that the CD117⁺ cell population includes endothelial precursors and can differentiate into endothelial cells in vitro.

Because we achieved only 20-fold expansion of CD117⁺ cells and found that the proliferation of CD117⁺ cells was very low after 2 wk of culture, we measured endogenous cellular -galactosidase activity, a marker of cellular senescence. More than 70% of cells were SA -galactosidase positive after 14 days of culture, indicating that the majority of these CD117⁺ stem cells had a senescent phenotype. Therefore, the proliferation of CD117⁺ cells was poor after 2 wk of culture. However, immunostaining analysis by TdT-mediated DUTP nickend labeling method showed that the apoptotic index was <1% in the expanded CD117⁺ cells after 2 wk of culture (data not shown).

To examine the angiogenic potency by the implantation of ex vivo expanded cells, we injected 2 x 10⁵ of either 2-wk expanded CD117⁺ cells or freshly collected CD117⁺ cells into the ischemic hindlimbs of mice. The cell survival and incorporation of implanted cells were obviously poorer when expanded CD117⁺ cells were used for implantation, rather than freshly collected CD117⁺ cells. Furthermore, we found that these ex vivo expanded cells have a low potential for inducing therapeutic angiogenesis, based on the assessment of the microvessel density and the percentage of limb blood flow in the ischemic hindlimbs 2 wk after treatment.

In contrast to other reports (13, 15), our results showed that the expanded CD117⁺ cells had a low potential for inducing therapeutic angiogenesis, relative to freshly isolated CD117⁺ cells, which could be attributed to the different cell source used for expansion or to the different culture conditions. However, we did not achieve satisfactory results even when we cultured the CD117⁺ cells under the same conditions as described by Kalka et al. (13).

Because the induction of therapeutic angiogenesis by cell therapy is related to both the production of angiogenic growth factors and the endothelial differentiation from implanted cells, several factors could compromise the angiogenic potential of expanded CD117⁺ cells. The decrease in angiogenic growth factor production is considered to be an important factor, although our previous investigation indicated the contrary because a high concentration of vascular endothelial growth factor was measured in the CD117⁺ cell suspension after 14 days of culture (19).

It is well known that the ex vivo expansion of adult stem cells is very difficult. The senescence or phenotype changes of ex vivo expanded CD117⁺ cells will result in decreased cell survival, endothelial differentiation, and incorporation after cell implantation, which is critically important for inducing angiogenesis. We speculate that the reason these expanded cells lost their potential for inducing angiogenesis was related to the senescence and the change of their phenotype, including the downregulation of membrane-bound receptors or adhesion molecules after ex vivo expansion. In fact, it was reported that ex vivo expanded CD34⁺ cells have poorer engraftment potential than unexpanded cells (29).

In summary, we found that the implantation of ex vivo expanded CD117⁺ cells has low potential for inducing therapeutic angiogenesis, which could be related to the senescence of these expanded cells, and to poor survival and incorporation after implantation into the ischemic hindlimbs of mice. Further studies are needed to develop a new culture system of cell supply by ex vivo expansion for potential clinical cell-based therapy.

	ACKNOWLEDGMENTS

 
We thank Mako Ohshima for excellent technical assistance.

GRANTS

This work was supported by Research Grant 13C-1 for Cardiovascular Diseases from the Ministry of Health, Labour and Welfare.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. Hamano, Div. of Cardiovascular Surgery, Dept. of Medical Bioregulation, Yamaguchi Univ. School of Medicine, Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan (E-mail: kimikazu{at}yamaguchi-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 

1. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, and Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964–967, 1997.[Abstract/Free Full Text]
2. Assmus B, Schachinger V, Teupe C, Britten M, Lehmann R, Dobert N, Grunwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, and Zeiher AM. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 106: 3009–3017, 2002.[Abstract/Free Full Text]
3. Bender JG, Unverzagt KL, Walker DE, Lee W, Van Epps DE, Smith DH, Stewart CC, and To LB. Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry. Blood 77: 2591–2596, 1991.[Abstract/Free Full Text]
4. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, Peacocke M, and Campisi J. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92: 9363–9367, 1995.[Abstract/Free Full Text]
5. Esato K, Hamano K, Li TS, Furutani A, Seyama A, Takenaka H, and Zempo N. Neovascularization induced by autologous bone marrow cell implantation in peripheral arterial disease. Cell Transplant 11: 747–752, 2002.[ISI][Medline]
6. Fuchs S, Satler LF, Kornowski R, Okubagzi P, Weisz G, Baffour R, Waksman R, Weissman NJ, Cerqueira M, Leon MB, and Epstein SE. Catheter-based autologous bone marrow myocardial injection in no-option patients with advanced coronary artery disease: a feasibility study. J Am Coll Cardiol 41: 1721–1724, 2003.[Abstract/Free Full Text]
7. Hamano K, Li TS, Kobayashi T, Kobayashi S, Matsuzaki M, and Esato K. Angiogenesis induced by the implantation of self-bone marrow cells: a new material for therapeutic angiogenesis. Cell Transplant 9: 439–443, 2000.[ISI][Medline]
8. Hamano K, Nishida M, Hirata K, Mikamo A, Li TS, Harada M, Miura T, Matsuzaki M, and Esato K. Local implantation of autologous bone marrow cells for therapeutic angiogenesis in patients with ischemic heart disease: clinical trial and preliminary results. Jpn Circ J 65: 845–847, 2001.[CrossRef][Medline]
9. Horvath KA, Cohn LH, Cooley DA, Crew JR, Frazier OH, Griffith BP, Kadipasaoglu K, Lansing A, Mannting F, March R, Mirhoseini MR, and Smith C. Transmyocardial laser revascularization: results of a multicenter trial with transmyocardial laser revascularization used as sole therapy for end-stage coronary artery disease. J Thorac Cardiovasc Surg 113: 645–653, 1997.[Abstract/Free Full Text]
10. Huss R. Isolation of primary and immortalized CD34-hematopoietic and mesenchymal stem cells from various sources. Stem Cells 18: 1–9, 2000.[Abstract/Free Full Text]
11. Isner JM and Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest 103: 1231–1236, 1999.[ISI][Medline]
12. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi KK, and Goodell MA. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 107: 1395–1402, 2001.[CrossRef][ISI][Medline]
13. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, and Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA 97: 3422–3427, 2000.[Abstract/Free Full Text]
14. Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, Masaki H, Mori Y, Iba O, Tateishi E, Kosaki A, Shintani S, Murohara T, Imaizumi T, and Iwasaka T. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 104: 1046–1052, 2001.[Abstract/Free Full Text]
15. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, and Asahara T. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 103: 634–637, 2001.[Abstract/Free Full Text]
16. Kobayashi T, Hamano K, Li TS, Katoh T, Kobayashi S, Matsuzaki M, and Esato K. Enhancement of angiogenesis by the implantation of self bone marrow cells in a rat ischemic heart model. J Surg Res 89: 189–195, 2000.[CrossRef][ISI][Medline]
17. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, and Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7: 430–436, 2001.[CrossRef][ISI][Medline]
18. Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, and Langer R. Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 99: 4391–4316, 2002.[Abstract/Free Full Text]
19. Li TS, Hamano K, Nishida M, Hayashi M, Ito H, Mikamo A, and Matsuzaki M. CD117⁺ stem cells play a key role in therapeutic angiogenesis induced by bone marrow cell implantation. Am J Physiol Heart Circ Physiol 285: H931–H937, 2003.[Abstract/Free Full Text]
20. Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD, Maysky M, Ashare AB, Lathi K, and Isner JM. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation 98: 2800–2804, 1998.[Abstract/Free Full Text]
21. Murohara T, Ikeda H, Duan J, Shintani S, Sasaki K, Eguchi H, Onitsuka I, Matsui K, and Imaizumi T. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 105: 1527–1536, 2000.[ISI][Medline]
22. Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, Mesquita CT, Rossi MI, Carvalho AC, Dutra HS, Dohmann HJ, Silva GV, Belem L, Vivacqua R, Rangel FO, Esporcatte R, Geng YJ, Vaughn WK, Assad JA, Mesquita ET, and Willerson JT. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 107: 2294–2302, 2003.[Abstract/Free Full Text]
23. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, and Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 109: 337–346, 2002.[CrossRef][ISI][Medline]
24. Schumacher B, Pecher P, von Specht BU, and Stegmann T. Induction of neoangiogenesis in ischemic myocardium by human growth factors: first clinical results of a new treatment of coronary heart disease. Circulation 97: 645–650, 1998.[Abstract/Free Full Text]
25. Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Klinge H, Schumichen C, Nienaber CA, Freund M, and Steinhoff G. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet 361: 45–46, 2003.[CrossRef][ISI][Medline]
26. Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, Kogler G, and Wernet P. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106: 1913–1918, 2002.[Abstract/Free Full Text]
27. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K, Akashi H, Shimada K, Iwasaka T, and Imaizumi T. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 360: 427–435, 2002.[CrossRef][ISI][Medline]
28. Tse HF, Kwong YL, Chan JK, Lo G, Ho CL, and Lau CP. Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet 361: 47–49, 2003.[CrossRef][ISI][Medline]
29. Xu R and Reems JA. Umbilical cord blood progeny cells that retain a CD34⁺ phenotype after ex vivo expansion have less engraftment potential than unexpanded CD34⁺ cells. Transfusion 41: 213–218, 2001.[CrossRef][ISI][Medline]

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