- Email: [email protected]
Human umbilical cord derived stem cells for the injured heart
Medical Hypotheses (2007) 68, 94–97
http://intl.elsevierhealth.com/journals/mehy
Human umbilical cord derived stem cells for the injured heart Kai Hong Wu a, Shao Guang Yang b, Bin Zhou b, Wei Ting Du b, Dong Sheng Gu b, Peng Xia Liu b, Wen Bin Liao b, Zhong Chao Han Ying Long Liu a,*
b,*
,
a
Pediatric Cardiac Center, Department of Surgery, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 167 Beilishi Road, Beijing 100037, China b State Key Laboratory of Experimental Hematology, National Research Center for Stem Cell Engineering and Technology, Institute of Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China Received 29 May 2006; accepted 2 June 2006
Summary The limited ability of the heart to regenerate damaged tissue following a myocardial infarct results in progressive dysfunctions and consequently leads to heart failure. Cell therapy with stem cells for cardiac repair is emerging as an alternative strategy and demonstrates promising results. Recent advances suggest human umbilical cord may be a new source for stem cells. Human umbilical cords are easy to obtain and umbilical cord derived stem cells can be easily extracted and cryopreserved, allowing for individuals to store their own samples for possible future autologous use even if there were no immediate indication that stem cell therapy would be required. Therefore, we hypothesize that human umbilical cord derived stem cells may be the new cell source for the injured heart. c 2006 Elsevier Ltd. All rights reserved.
Introduction The limited ability of the heart to regenerate damaged tissue following a myocardial infarct results in progressive dysfunctions and consequently leads to heart failure. There is growing
* Corresponding author. Tel.: +86 10 88398188; fax: +86 10 68332747. E-mail addresses: [email protected] (Z.C. Han), [email protected] (Y.L. Liu).
enthusiasm for the application of stem cells to repair or regenerate damaged myocardium [1,2]. Recent advances suggest human umbilical cord (hUC) may be a new source for stem cells [3]. In our laboratory, we established a simple method to isolate stem cells from hUC tissues, called hUC-derived stem (UCDS) cells. These cells display a fibroblast-like morphology, express mesenchymal markers, and have the potential to differentiate into osteogenic, adipogenic, and cardiogenic cells. Therefore, UCDS cells may be the new cell source for the injured heart.
0306-9877/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2006.06.038
Human umbilical cord derived stem cells for the injured heart
Materials and methods Fresh umbilical cords were collected from normal full-term pregnancies and the cells were isolated immediately. In brief, the hUC tissues were minced into 1–2 mm3 small fragments with scissors, and enzymatically digested with 0.075% collagenase type II and 0.125% trypsin in PBS with gentle agitation at 37 C for 30 min. The digested mixture was then passed through a 100 lm filter to obtain cell suspensions. The dissociated cells were centrifuged at 2000 rpm for 18 min at room temperature and washed 3 times. Cells were then plated in growth medium with a concentration of 1 · 106 cells/cm2
95
and allowed to proliferate in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, DF12), supplemented with 10% fetal bovine serum (FBS), 10 ng/ ml epidermal growth factor (EGF). Medium was replaced at 48 h and every 2 or 3 days following. The cells were serially passaged and expanded in a humidified incubator at 37 C with 5% CO2. The cell surface markers of UCDS cells were analyzed by flow cytometry. Osteogenic differentiation of UCDS cells was induced by application to osteogenic medium, which contained dexamethasone, b-glycerolphosphate, and ascorbic acid. For adipogenic differentiation, the UCDS cells were induced in adipogenic medium containing hydrocortisone,
Figure 1 Morphology and differentiation ability of UCDS cells. (A) Morphology of primary cultured UCDS cells. (B) Morphology of sub-cultured UCDS cells, the cells were fibroblast-like and grew as a whirlpool. (C) Von Kossa staining of the UCDS cells after osteogenic induction. (D) Oil-red O staining of the UCDS cells after adipogenic induction. DAB staining of cardiac sarcomeric a-actin and Troponin T at 1 month after 5-azacytidine treatment (E, F).
96 isobutylmethylxanthine, and indomethacin. After 2 weeks of induction, the cells were stained with von Kossa procedure or Oil red solution to detect the presence of calcium deposition in osteocytes or neutral lipid vacuoles in adipocytes, respectively. For cardiomyogenic differentiation, the cells were incubated in DF12 supplemented with 10% FBS, 5 lg/mL insulin and EGF (10 ng/ml). Four hours after seeding, the cultures were treated with 5-azacytidine (10 lmol/L) for 24 h, then the medium was changed and the cells were maintained in the medium for 3 to 5 weeks. Immunocytochemistry was performed as described previously [4] to test the expression of cardiac specific protein, such ascardiac sarcomeric a-actin and Troponin T.
Results
Wu et al. Table 1 Flow cytometry results of UCDS cells labeled for cell-surface markers compared with BMMSCs: , negative <2%; +, positive >90%; ++, strongly positive, >98% Surface marker
BM-MSCs
UCDS cells
MHC-I MHC-II CD13 CD29 CD38 CD31 CD44 CD45 CD90 CD105 CD106 CD117 CD166
88.44
35.23
++ ++
+ ++
++
++
+ + 8.74
+ + 3.16
63.35
85.42
Data are representative of several independent experiments.
The UCDS cells isolated in our laboratory showed heterogeneity during the first 3 days. When initially plated, the UCDS cells appeared rounded in shape. After 48 or 72 h of plating, the cells were adherent, elongated, and spindle-shaped (Fig. 1A). When the medium was changed, the suspending cells were removed and the sub-cultured cells were much pure and fibroblast-like (Fig. 1B). The primary culture cells reached confluence about 1 or 2 weeks later, the cells sub-cultured at a ratio of 1:3 reached confluence 2 or 3 days later. When UCDS cells were cultured in osteogenic medium for 2 weeks, the morphology changed, began to mineralize their matrix and was positive for Kossa staining (Fig. 1C). They were also able to differentiate into adipocytes, and cells accumulated different amounts of lipid vacuoles after cultivation in adipogenic medium (Fig. 1D). Immunocytochemistry showed that differentiated cells were strongly stained with cardiac sarcomeric a-actin and Troponin T at 1 month after 5-azacytidine treatment (Fig. 1E and F). Also, the UCDS cells can be differentiated into hepatogenic, endothelial and neural cells in vitro (data not shown). Flow cytometry results showed that UCDS cells expressed CD13, CD29, CD44, CD90, CD105, and CD166 but not CD38, CD45, CD106, and MHC class II, and show fractional expression of the marker CD166, MHC class I similar to that of bone marrow (BM) derived mesenchymal stem cells (MSCs) (Table 1). The UCDS cells appeared morphologically to be a homogeneous population, maintained similar morphology with passages and could be passaged more than 20 times without detecting signs of senescence. These results suggest that the UCDS cells are a crowd of undifferentiated stem cells that are different from hematopoietic stem cells.
Discussion and hypothesis Stem cells are unspecialized precursor cells that are defined by their capacity for self-renewal through symmetric division. They can remain undifferentiated and divide for long periods of time, or they can develop into specialized cell lineages [5]. Adult MSCs have shown great promise in cell therapy applications. Transplantation of MSCs in an animal model has been shown to improve the functioning of the infarcted heart through myogenesis and angiogenesis [6]. The therapeutic potential of MSCs in cardiovascular disease is significant, however, MSCs are rare in adult human bone marrow and the number or plasticity of stem cell populations significantly decreases with age or age-related disease. Also, obtaining the therapeutic quantity of bone marrow requires anesthesia and hospitalization. In addition, high degree of viral infection in patients and the ethical issues surrounding embryonic stem cells which make it necessary to search for alternative sources of these cells for autologous and allogenic use [7,8]. The hUC vessels and surrounding mesenchyme are derived from extraembyronic mesoderm and/ or embryonic mesoderm. Thus, these tissues are rich sources of stem cells that may be useful for a variety of therapeutic purposes. In the present study, we develop a method that can readily isolate and expand stem cells from hUC tissues, these cells can be differentiated into osteogenic, adipogenic and cardiomyogenic cells in vitro. FACS analysis showed that the cell surface makers of UCDS cells were similar to that of bone marrow derived
Human umbilical cord derived stem cells for the injured heart MSCs. Special attention should be paid to the expression of MHC molecules. Since MHC usually mediates the allogenic response, the absence of MHC-II molecules and low expression of MHC-I molecules on UCDS cells may have significant implications in that UCDS cells may be used for allogenic cell transplantation with lower risk of alloreactivity. Thus, UCDS cells represent an attractive autologous cell source for cell therapy. Interestingly, hUC derived cells have been used in cardiovascular tissue engineering in an experiment and good results in this field have been achieved [9]. Consequently, we hypothesize that UCDS cells may be the new cell source for the injured heart.
Conclusion In summary, UCDS cells can be successfully isolated from hUC tissues. The unique capabilities of these cells for pluripotency and long-term self-renewal make it an ideal source for the regeneration of injured heart and the degenerative diseases of all organ systems. These cells can be easily extracted and cryopreserved, allowing for individuals to store their own samples for possible future autologous use even if there were no immediate indication that stem cell therapy would be required. We believe in the near future, cryopreserved autologous UCDS cells for the injured heart may become available.
Ethical approval Tissue collection for research was approved by the institutional review board of the Chinese Academy
97
of Medical Science and Peking Union Medical College.
Acknowledgement This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education (20040023048) from the Ministry of Education of China.
References [1] Wollert KC, Drexler H. Clinical applications of stem cells for the heart. Circ Res 2005;96:151–63. [2] Wu K, Liu YL, Cui B, Han Z. Application of stem cells for cardiovascular grafts tissue engineering. Transpl Immunol 2006;16:1–7. [3] Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 2004;22:1330–7. [4] Ding S, Merkulova-Rainon T, Han ZC, Tobelem G. HGF receptor up-regulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro. Blood 2003;101:4816–22. [5] Zavos PM. Stem cells and cellular therapy: potential treatment for cardiovascular diseases. Int J Cardiol 2006;107:1–6. [6] Olivares EL, Ribeiro VP, Werneck de Castro JP, et al. Bone marrow stromal cells improve cardiac performance in healed infarcted rat hearts. Am J Physiol Heart Circ Physiol 2004;287:H464–70. [7] Zhang H, Fazel S, Tian H, et al. Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy. Am J Physiol Heart Circ Physiol 2005;289:H2089–96. [8] McLaren A. Ethical and social considerations of stem cell research. Nature 2001;414:129–31. [9] Kadner A, Hoerstrup SP, Tracy J, et al. Human umbilical cord cells: a new cell source for cardiovascular tissue engineering. Ann Thorac Surg 2002;74:S1422–8.
http://intl.elsevierhealth.com/journals/mehy
Human umbilical cord derived stem cells for the injured heart Kai Hong Wu a, Shao Guang Yang b, Bin Zhou b, Wei Ting Du b, Dong Sheng Gu b, Peng Xia Liu b, Wen Bin Liao b, Zhong Chao Han Ying Long Liu a,*
b,*
,
a
Pediatric Cardiac Center, Department of Surgery, Cardiovascular Institute and Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 167 Beilishi Road, Beijing 100037, China b State Key Laboratory of Experimental Hematology, National Research Center for Stem Cell Engineering and Technology, Institute of Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China Received 29 May 2006; accepted 2 June 2006
Summary The limited ability of the heart to regenerate damaged tissue following a myocardial infarct results in progressive dysfunctions and consequently leads to heart failure. Cell therapy with stem cells for cardiac repair is emerging as an alternative strategy and demonstrates promising results. Recent advances suggest human umbilical cord may be a new source for stem cells. Human umbilical cords are easy to obtain and umbilical cord derived stem cells can be easily extracted and cryopreserved, allowing for individuals to store their own samples for possible future autologous use even if there were no immediate indication that stem cell therapy would be required. Therefore, we hypothesize that human umbilical cord derived stem cells may be the new cell source for the injured heart. c 2006 Elsevier Ltd. All rights reserved.
Introduction The limited ability of the heart to regenerate damaged tissue following a myocardial infarct results in progressive dysfunctions and consequently leads to heart failure. There is growing
* Corresponding author. Tel.: +86 10 88398188; fax: +86 10 68332747. E-mail addresses: [email protected] (Z.C. Han), [email protected] (Y.L. Liu).
enthusiasm for the application of stem cells to repair or regenerate damaged myocardium [1,2]. Recent advances suggest human umbilical cord (hUC) may be a new source for stem cells [3]. In our laboratory, we established a simple method to isolate stem cells from hUC tissues, called hUC-derived stem (UCDS) cells. These cells display a fibroblast-like morphology, express mesenchymal markers, and have the potential to differentiate into osteogenic, adipogenic, and cardiogenic cells. Therefore, UCDS cells may be the new cell source for the injured heart.
0306-9877/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2006.06.038
Human umbilical cord derived stem cells for the injured heart
Materials and methods Fresh umbilical cords were collected from normal full-term pregnancies and the cells were isolated immediately. In brief, the hUC tissues were minced into 1–2 mm3 small fragments with scissors, and enzymatically digested with 0.075% collagenase type II and 0.125% trypsin in PBS with gentle agitation at 37 C for 30 min. The digested mixture was then passed through a 100 lm filter to obtain cell suspensions. The dissociated cells were centrifuged at 2000 rpm for 18 min at room temperature and washed 3 times. Cells were then plated in growth medium with a concentration of 1 · 106 cells/cm2
95
and allowed to proliferate in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, DF12), supplemented with 10% fetal bovine serum (FBS), 10 ng/ ml epidermal growth factor (EGF). Medium was replaced at 48 h and every 2 or 3 days following. The cells were serially passaged and expanded in a humidified incubator at 37 C with 5% CO2. The cell surface markers of UCDS cells were analyzed by flow cytometry. Osteogenic differentiation of UCDS cells was induced by application to osteogenic medium, which contained dexamethasone, b-glycerolphosphate, and ascorbic acid. For adipogenic differentiation, the UCDS cells were induced in adipogenic medium containing hydrocortisone,
Figure 1 Morphology and differentiation ability of UCDS cells. (A) Morphology of primary cultured UCDS cells. (B) Morphology of sub-cultured UCDS cells, the cells were fibroblast-like and grew as a whirlpool. (C) Von Kossa staining of the UCDS cells after osteogenic induction. (D) Oil-red O staining of the UCDS cells after adipogenic induction. DAB staining of cardiac sarcomeric a-actin and Troponin T at 1 month after 5-azacytidine treatment (E, F).
96 isobutylmethylxanthine, and indomethacin. After 2 weeks of induction, the cells were stained with von Kossa procedure or Oil red solution to detect the presence of calcium deposition in osteocytes or neutral lipid vacuoles in adipocytes, respectively. For cardiomyogenic differentiation, the cells were incubated in DF12 supplemented with 10% FBS, 5 lg/mL insulin and EGF (10 ng/ml). Four hours after seeding, the cultures were treated with 5-azacytidine (10 lmol/L) for 24 h, then the medium was changed and the cells were maintained in the medium for 3 to 5 weeks. Immunocytochemistry was performed as described previously [4] to test the expression of cardiac specific protein, such ascardiac sarcomeric a-actin and Troponin T.
Results
Wu et al. Table 1 Flow cytometry results of UCDS cells labeled for cell-surface markers compared with BMMSCs: , negative <2%; +, positive >90%; ++, strongly positive, >98% Surface marker
BM-MSCs
UCDS cells
MHC-I MHC-II CD13 CD29 CD38 CD31 CD44 CD45 CD90 CD105 CD106 CD117 CD166
88.44
35.23
++ ++
+ ++
++
++
+ + 8.74
+ + 3.16
63.35
85.42
Data are representative of several independent experiments.
The UCDS cells isolated in our laboratory showed heterogeneity during the first 3 days. When initially plated, the UCDS cells appeared rounded in shape. After 48 or 72 h of plating, the cells were adherent, elongated, and spindle-shaped (Fig. 1A). When the medium was changed, the suspending cells were removed and the sub-cultured cells were much pure and fibroblast-like (Fig. 1B). The primary culture cells reached confluence about 1 or 2 weeks later, the cells sub-cultured at a ratio of 1:3 reached confluence 2 or 3 days later. When UCDS cells were cultured in osteogenic medium for 2 weeks, the morphology changed, began to mineralize their matrix and was positive for Kossa staining (Fig. 1C). They were also able to differentiate into adipocytes, and cells accumulated different amounts of lipid vacuoles after cultivation in adipogenic medium (Fig. 1D). Immunocytochemistry showed that differentiated cells were strongly stained with cardiac sarcomeric a-actin and Troponin T at 1 month after 5-azacytidine treatment (Fig. 1E and F). Also, the UCDS cells can be differentiated into hepatogenic, endothelial and neural cells in vitro (data not shown). Flow cytometry results showed that UCDS cells expressed CD13, CD29, CD44, CD90, CD105, and CD166 but not CD38, CD45, CD106, and MHC class II, and show fractional expression of the marker CD166, MHC class I similar to that of bone marrow (BM) derived mesenchymal stem cells (MSCs) (Table 1). The UCDS cells appeared morphologically to be a homogeneous population, maintained similar morphology with passages and could be passaged more than 20 times without detecting signs of senescence. These results suggest that the UCDS cells are a crowd of undifferentiated stem cells that are different from hematopoietic stem cells.
Discussion and hypothesis Stem cells are unspecialized precursor cells that are defined by their capacity for self-renewal through symmetric division. They can remain undifferentiated and divide for long periods of time, or they can develop into specialized cell lineages [5]. Adult MSCs have shown great promise in cell therapy applications. Transplantation of MSCs in an animal model has been shown to improve the functioning of the infarcted heart through myogenesis and angiogenesis [6]. The therapeutic potential of MSCs in cardiovascular disease is significant, however, MSCs are rare in adult human bone marrow and the number or plasticity of stem cell populations significantly decreases with age or age-related disease. Also, obtaining the therapeutic quantity of bone marrow requires anesthesia and hospitalization. In addition, high degree of viral infection in patients and the ethical issues surrounding embryonic stem cells which make it necessary to search for alternative sources of these cells for autologous and allogenic use [7,8]. The hUC vessels and surrounding mesenchyme are derived from extraembyronic mesoderm and/ or embryonic mesoderm. Thus, these tissues are rich sources of stem cells that may be useful for a variety of therapeutic purposes. In the present study, we develop a method that can readily isolate and expand stem cells from hUC tissues, these cells can be differentiated into osteogenic, adipogenic and cardiomyogenic cells in vitro. FACS analysis showed that the cell surface makers of UCDS cells were similar to that of bone marrow derived
Human umbilical cord derived stem cells for the injured heart MSCs. Special attention should be paid to the expression of MHC molecules. Since MHC usually mediates the allogenic response, the absence of MHC-II molecules and low expression of MHC-I molecules on UCDS cells may have significant implications in that UCDS cells may be used for allogenic cell transplantation with lower risk of alloreactivity. Thus, UCDS cells represent an attractive autologous cell source for cell therapy. Interestingly, hUC derived cells have been used in cardiovascular tissue engineering in an experiment and good results in this field have been achieved [9]. Consequently, we hypothesize that UCDS cells may be the new cell source for the injured heart.
Conclusion In summary, UCDS cells can be successfully isolated from hUC tissues. The unique capabilities of these cells for pluripotency and long-term self-renewal make it an ideal source for the regeneration of injured heart and the degenerative diseases of all organ systems. These cells can be easily extracted and cryopreserved, allowing for individuals to store their own samples for possible future autologous use even if there were no immediate indication that stem cell therapy would be required. We believe in the near future, cryopreserved autologous UCDS cells for the injured heart may become available.
Ethical approval Tissue collection for research was approved by the institutional review board of the Chinese Academy
97
of Medical Science and Peking Union Medical College.
Acknowledgement This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education (20040023048) from the Ministry of Education of China.
References [1] Wollert KC, Drexler H. Clinical applications of stem cells for the heart. Circ Res 2005;96:151–63. [2] Wu K, Liu YL, Cui B, Han Z. Application of stem cells for cardiovascular grafts tissue engineering. Transpl Immunol 2006;16:1–7. [3] Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 2004;22:1330–7. [4] Ding S, Merkulova-Rainon T, Han ZC, Tobelem G. HGF receptor up-regulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro. Blood 2003;101:4816–22. [5] Zavos PM. Stem cells and cellular therapy: potential treatment for cardiovascular diseases. Int J Cardiol 2006;107:1–6. [6] Olivares EL, Ribeiro VP, Werneck de Castro JP, et al. Bone marrow stromal cells improve cardiac performance in healed infarcted rat hearts. Am J Physiol Heart Circ Physiol 2004;287:H464–70. [7] Zhang H, Fazel S, Tian H, et al. Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy. Am J Physiol Heart Circ Physiol 2005;289:H2089–96. [8] McLaren A. Ethical and social considerations of stem cell research. Nature 2001;414:129–31. [9] Kadner A, Hoerstrup SP, Tracy J, et al. Human umbilical cord cells: a new cell source for cardiovascular tissue engineering. Ann Thorac Surg 2002;74:S1422–8.