Cotransplantation of human umbilical cord-derived mesenchymal stem cells and umbilical cord blood-derived CD34+ cells in a rabbit model of myocardial infarction
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- Li, T., Ma, Q., Ning, M. et al. Mol Cell Biochem (2014) 387: 91. doi:10.1007/s11010-013-1874-5
The objective of the study is to investigate the effect of hypoxic preconditioning on the immunomodulatory properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and the effect of cotransplantation of hUC-MSCs and human umbilical cord blood (hUCB)-derived CD34+ cells in a rabbit model of myocardial infarction. hUC-MSCs with or without hypoxic preconditioning by cobalt chloride were plated in a 24-well plate, and then cocultured with hUCB-CD34+ cells and PBMCs for 96 h at 37 °C in a 5 % CO2 incubator. For the negative control, hUC-MSCs were omitted. The groups were divided as follows: A1 = HP-MSCs + hUCB-CD34+ cells + PBMC, A2 = hUC-MSCs + hUCB-CD34+ cells + PBMC, Negative Control = hUCB-CD34+ cells + PBMC. Culture supernatants of each group were collected, and the IL-10 and IFN-γ levels were measured by ELISA. A rabbit model of MI was established using a modified Fujita method. The animals were then randomized into three groups and received intramyocardial injections of 0.4 ml of PBS alone (n = 8, PBS group), hUC-MSCs in PBS (n = 8, hUC-MSCs group), or hUC-MSCs + CD34+ cells in PBS (n = 8, Cotrans group), at four points in the infarct border zone. Echocardiography was performed at baseline, 4 weeks after MI induction, and 4 weeks after cell transplantation, respectively. Stem cell differentiation and neovascularization in the infracted area were characterized for the presence of cardiac Troponin I (cTnI) and CD31 by immunohistochemical staining, and the extent of myocardial fibrosis was evaluated by hematoxylin and eosin (H&E) and Masson’s trichrome. IFN-γ was 27.00 ± 1.11, 14.20 ± 0.81, and 7.22 ± 0.14 pg/ml, and IL-10 was 31.68 ± 3.08, 61.42 ± 1.08, and 85.85 ± 1.80 pg/ml for the Control, A1 and A2 groups, respectively, which indicated that hUCB-CD34+ cells induced immune reaction of peripheral blood mononuclear cells, whereas both hUC-MSCs and HP-MSCs showed an immunosuppressive effect, which, however, was attenuated by hypoxic preconditioning. The Cotrans group had less collagen deposition in the infarcted myocardium and better heart function than the hUC-MSCs or PBS group. The presence of cTnI-positive cells and CD31-positive tubular structures indicated the differentiation of stem cells into cardiomyocytes and neovascularization. The microvessel density was 12.19 ± 3.05/HP for the hUC-MSCs group and 31.63 ± 2.45/HP for the Cotrans group, respectively (P < 0.01). As a conclusion, both hUC-MSCs and HP-MSCs have an immunosuppressive effect on lymphocytes, which, however, can be attenuated by hypoxic preconditioning. Cotransplantation of hUC-MSCs and hUCB-CD34+ cells can improve heart function and decrease collagen deposition in post-MI rabbits. Thus, a combined regimen of hUC-MSCs and hUCB-CD34+ cells would be more desirable than either cells administered alone. This is most likely due to the increase of cardiomyocytes and enhanced angiogenesis in the infarcted myocardium.
KeywordsMyocardial InfarctionUmbilical cord mesenchymal stem cellsUmbilical cord blood-derived CD34+ CellsCotransplantation
Acute myocardial infarction (AMI) promotes an irreversible and massive loss of cardiomyocytes, followed by gradual replacement of these damaged cardiomyocytes with fibrous non-contractile cells and eventually heart failure [1, 2]. Cellular cardiomyoplasty holds great promise for the repair or regeneration of infarcted myocardium, in which exogenous stem cells, such as umbilical cord-derived mesenchymal stem cells (UC-MSCs) [3, 4] and peripheral blood/umbilical cord blood (PB/UCB)-derived CD34+ cells [5, 6], are injected into the damaged myocardium.
Bone marrow (BM) represents the most widely used source of allogeneic MSCs, it is, however, limited by the availability of donors because BM aspiration is painful and may pose risks and complications to some donors. Umbilical cord matrix or Wharton’s jelly has been suggested as an alternative source of MSCs for the repair and regeneration of the infarcted or ischemic cardiovascular tissues [4, 7]. The frequency of hematopoietic stem cells and progenitor cells in UCB equals or even exceeds that of BM, and human umbilical cord blood (hUCB) contains up to tenfold higher amounts of CD34+ endothelial precursor cells as non-mobilized adult peripheral blood [8, 9]. Several animal studies have shown that CD34+ cells could differentiate into vascular endothelial cells that contribute to the increase in the number of microvessels and improvement of heart function [6, 10].
MSCs are known to improve heart function via angiogenesis induced by pro-angiogenic factors, the effect of which can be increased by hypoxic preconditioning . Zhou et al.  and Weiss et al.  have also shown that UC-MSCs have low immunogenicity and immunomodulatory properties. A question arises whether these immunomodulatory properties are retained in hypoxic preconditioned UC-MSCs, which will be addressed in this study.
Despite their therapeutic potential and advantages compared with BM-MSCs, there have been few studies on the use of UC-MSCs and UCB-CD34+ cells , and to our knowledge no studies about the cotransplantation of UC-MSCs and UCB-CD34+ for the treatment of MI. In line with previous findings, it is hypothesized in this study that cotransplantation of UC-MSCs and UCB-CD34+ might have a better effect than either cells administered alone in post-MI animals.
Materials and methods
The study protocol was approved by the Institutional Review Board of Tianjin Medical University and the Human Research Ethics Committee of Tianjin Third Central Hospital. All participants provided written informed consent, and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.
Isolation and culture of human UC-MSCs
Human umbilical cords were collected from consenting mothers in the maternity ward of our hospital. They were exhaustively washed with PBS to remove residual blood clots and blood vessels, minced into small pieces of approximately 1–2 mm3 in size, and then incubated with 0.1 % type IV collagenase (GIBCO, USA) for 60 min. After centrifugation and washing with PBS, the tissues were resuspended in low-glucose DMEM/F12 (Bioroc, Tianjin, China) supplemented with 10 % fetal bovine serum (FBS, GIBCO, USA) and 100,000 U/ml of penicillin/streptomycin, and then cultured in a humidified 5 % CO2 incubator at 37 °C.
Isolation and culture of peripheral blood mononuclear cells (PBMCs)
Human PBMCs were isolated from the peripheral blood of health donors by Ficoll Histopaque (1.077 g/ml) density gradient centrifugation (MD Pacific, Tianjin, China), and the cell concentration was adjusted to 1 × 106/ml with RPMI 1640 medium (GIBCO, USA).
Isolation and culture of hUCB-CD34+ cells
hUCB was also obtained from the mothers. Red cells were removed by sedimentation in 6 % hydroxyethyl starch (HES, Fresenius Kabi, Germany), and then the mononuclear cells were isolated from hUCB by a density gradient centrifugation method, from which the CD34 cells were positively selected by immunomagnetic bead separation using a human CD34 Microbead kit (Miltenyi Biotec, Germany). The selected CD34+ cells were plated in a T-25 culture flask in the STEMPRO®-34 SFM complete medium (GIBCO, USA).
Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) (2 × 105) in the third passage (P3) were trypsinized, suspended in 200 μl PBS, and then incubated for 30 min at room temperature with PE- or FITC-conjugated mouse anti-human monoclonal antibodies (CD34, CD45, CD90, and CD105). Mouse isotype antibodies served as controls. The resuspended cells were washed and then subjected to flow cytometry (FACSort, B-D Co., USA). The purity of the isolated CD34+ cells was also detected by flow cytometry.
Hypoxic preconditioning of hUC-MSCs
P3 hUC-MSCs were incubated in DMEM/F12 medium containing 100 μmol/l of cobalt chloride and 0.1 % FBS in a humidified 5 % CO2 incubator at 37 °C for 48 h.
P3 hUC-MSCs with or without hypoxic preconditioning were trypsinized, counted, and plated in a 24-well plate at a density of 2 × 104 per well, with six replicate wells for each group. After adherence of MSCs to the wall surface, mitomycin-C of 25 μg/ml (MMC, Kyowa Hakko Kogyo, Japan) was added into each well to mitotically inactivate MSCs. Then hUCB-CD34+ cells (2 × 104/well) and PBMCs (2 × 105/well) suspended in RPMI-1640 were added and cultured for 96 h at 37 °C in a 5 % CO2 incubator. For the negative control, hUC-MSCs were omitted. The groups were divided as follows: A1 = HP-MSCs + hUCB-CD34+ cells + PBMC, A2 = hUC-MSCs + hUCB-CD34+ cells + PBMC, Negative Control = hUCB-CD34+ cells + PBMC. Culture supernatants of each group were collected, and the IL-10 and IFN-γ levels were measured with a ELISA detection kit (Ever, USA). Each well was repeated twice following the manufacturer’s instructions.
A rabbit model of MI was established using a modified Fujita method . Adult female Japanese white rabbits, weighing 2.57 ± 0.45 kg, were anesthetized by intramuscular injection of ketamine (25 mg/kg) and intraperitoneal injection of 1 % pentobarbital sodium (1 ml/kg). A median incision was made, and the left ventricular branch (LVB) was ligated at the midpoint between the starting point of the major branch and the cardiac apex with a 6–0 Prolene suture. Myocardial ischemia was confirmed by both ST-segment elevation on the ECG and regional cyanosis of the myocardial surface. No drainage was performed. The animals were kept warm with a heating pad and allowed to recover. The survived rabbits were administered with 80 mg/kg penicillin im QD for 3 days.
A second thoracotomy was performed 4 weeks after MI following the same procedures as described above. The animals were randomized into three groups and received intramyocardial injections of 0.4 ml of PBS alone (n = 8, PBS group), 5 × 106 hUC-MSCs in PBS (n = 8, hUC-MSCs group), or 5 × 106 hUC-MSCs + 5 × 105/kg CD34+ cells in PBS (n = 8, Cotrans group), at four points in the infarct border zone.
Evaluation of heart function
Echocardiography was performed at baseline, 4 weeks after MI induction, and 4 weeks after cell transplantation, respectively.
The rabbits were euthanized by 10 % KCl f4 weeks after cell transplantation. The hearts were excised, fixed in 10 % formalin for >24 h, and cut transversely at the ligation. Then the myocardial tissues below the ligation site were embedded in paraffin and sectioned into 4- to 5-μm-thick slices, which were to be used for hematoxylin and eosin (H&E), Masson’s trichrome, and immunohistochemistry.
For the immunohistochemical detection of CD31, the tissue sections were incubated with the primary mouse monoclonal antibody to CD31 (1:15, Abcam, UK), followed by a second incubation with HRP-conjugated goat anti-mouse IgG antibody (Two-Step IHC Detection Reagent, ZSGB-BIO, China). For the detection of cTnI, the sections were incubated with the sheep polyclonal anti-cTnI antibody (1:100, Abcam, UK) and then HRP-conjugated rabbit anti-sheep IgG secondary antibody (1:500, CUSABIO, China). At last, the tissue sections were stained with DAB.
Determination of vessel density
CD31-positive vessels were counted in five randomly selected high-power fields under a light microscope at 200× (Olympus, Japan), and the vessel density was defined as the mean number blood of vessels.
All data were expressed as mean ± SE. Statistical analysis was performed by one-way ANOVA, followed by LSD post hoc test using SPSS version 19.0 (SPSS Inc., USA). P < 0.05 was considered statistically significant.
Isolation and culture of hUC-MSCs
Immunophenotype of hUC-MSCs and purity of CD34+ cells
Immunomodulatory effects of hUC-MSCs and HP-MSCs
14.20 ± 0.81*,#
61.42 ± 1.08*,#
7.22 ± 0.14*
85.85 ± 1.80*
27.00 ± 1.11
31.68 ± 3.08
Pathological changes of infarcted myocardium
The presence of CD31-positive tubular structures in the peri-infarcted area could be interpreted as an indicator of neovascularization in animals treated with stem cells (Fig. 6). The microvessel density was 12.19 ± 3.05/HP for the hUC-MSCs group and 31.63 ± 2.45/HP for the Cotrans group, respectively (P = 0.000). However, no CD31-positive vessels were detected in the PBS group.
In this study we showed that transplantation of hUC-MSCs or hUCB-CD34+ cells improved heart function in post-MI rabbits, and that PBS-treated animals had a persistently depressed left ventricular function. However, a combined regimen of hUC-MSCs and hUCB-CD34+ cells would be more desirable than either cells alone. Clearly, our results have important implications for stem cell-based therapy for MI. In this study, MSCs were successfully isolated from UC by enzyme digestion, and CD34+ cells with a high level of purity were positively selected from UCB by immunomagnetic bead separation. The ELISA results indicated that peripheral blood lymphocytes could be activated by UCB-CD34+ cells to secrete IFN-γ, which could modulate cell-mediated immunity and immune rejection. However, a decreased IFN-γ secretion and increased IL-10 secretion were observed in rabbits cocultured with hUC-MSCs. IL-10 has been reported to downregulate CD80 expression, disable T cells, and induce immune tolerance . The present study showed that cotransplantation of hUC-MSCs and hUCB-CD34+ cells resulted in an improved immunological tolerance of cardiomyocytes.
hUC-MSCs do not express HLA-class-II molecules and express only a low level of HLA-class-I molecules , indicating that hUC-MSCs have a low immunogenicity and are immunoprivileged. In addition, hUC-MSCs do not express costimulatory molecules such as CD40, CD80, and CD86 and thus are unable to stimulate the proliferation of human peripheral blood lymphocytes . hUC-MSCs secrete no IFN-γ and little IL-10 (<7.8 pg/ml) [10, 17]. All these results indicate that hUC-MSCs have immunosuppressive properties. Thus, from an immunological perspective, it makes possible cotransplantation of hUC-MSCs and hUCB-CD34+ for the treatment of MI.
The ELISA results showed that the immunosuppressive effect of the hypoxia-preconditioned hUC-MSCs was attenuated, the underlying mechanism remains to be determined. In this regard, despite an enhanced secretion of pro-angiogenic factors in response to hypoxic preconditioning , cotransplantation of HP-MSCs and hUCB-CD34+ cells might not be a good choice for the treatment of MI.
Several animal studies have shown that BM-MSCs could restore heart function after MI, decrease collagen deposition, and ameliorate LV remodeling [2, 11, 18]. Nevertheless, there is a paucity of studies on the treatment of MI with hUC-MSCs. Latifpour et al.  showed that undifferentiated hUC-MSCs improved heart function after MI and differentiated into cardiomyocytes in vitro. Our results also showed that hUC-MSCs improved heart function after MI, with an increase of LVFS from 31.25 ± 2.12 to 36.25 ± 1.75 % and less collagen deposition. Cotransplantation of hUC-MSCs and hUCB-CD34+ cells resulted in a higher LVFS and improved heart function as compared with hUC-MSCs administered alone.
There has been an ongoing debate about the mechanisms responsible for stem cell therapy for MI. It has been proved that stem cells differentiate into cardiomyocytes in vitro and in vivo [4, 19], but with an extremely low efficiency . In this study, immunohistochemical staining revealed the presence of a great number of cTnI-positive cells in the infarcted area of stem cell-treated animals. It is most likely that hUC-MSCs interact with hUCB-CD34+ cells that enhances the transdifferentiation of stem cells to cardiomyocytes. The exact mechanism that accounts for this is still unknown, but it might be related to cytokines secreted by MSCs that prevent early death of the stem cells and promote their survival and proliferation. Williams et al.  also showed that combining human cardiac stem cells with hMSCs produced a greater infarct size reduction and improved heart function as compared with either cells administered alone, and it showed sevenfold enhanced engraftment of stem cells in the combination therapy group versus either cell type alone.
It is also argued that stem cells differentiate into endothelial cells, resulting in an increase of vessel density in the infarcted area, tissue reperfusion, and eventually improved heart function [22, 23]. We found that there were more CD31-positive microvessels in the Cotrans group than in the hUC-MSCs group, suggesting that cotransplantation of hUC-CD34+ cells and hUC-MSCs has the potential to increase neovascularization. Again, this is more likely due to soluble cytokines secreted by stem cells, but not due to differentiation of stem cells. Nevertheless, a more rigorous test of this hypothesis is needed before a solid conclusion can be drawn. MSCs expressed higher vascular endothelial growth factor (VEGF) mRNA than hemopoietic progenitor cells in BM , and the expression of VEGF and basic fibroblast growth factor in the heart tissues of a swine model of chronic MI was increased after infusion of BM-MSCs . Thus it is believed that paracrine function may constitute the primary mechanism responsible for the stem cell therapy for MI.
This study has important theoretical and applied implications for stem cell therapy in post-MI patients. Both hUCB-CD34+ cells and hUC-MSCs are easily accessible without any invasive procedures and ethical problems. In addition, mesenchymal stem cells have an immunosuppressive ability so that immuno-suppressant is not necessary. hUC-MSCs have multipotency of differentiation into various tissue cells, including chondrocytes , adipocytes , and osteoblasts , and are, therefore, an ideal candidate for cellular therapy. They have a shorter population doubling time than BM-MSCs . Recent advances of stem cell biology make possible a more favorable therapeutic outcome with the use of complementary cells.
Both hUC-MSCs and HP-MSCs have an immunosuppressive effect on lymphocytes, which, however, can be attenuated by hypoxic preconditioning. Cotransplantation of hUC-MSCs and hUCB-CD34+ cells can improve heart function and decrease collagen deposition in post-MI rabbits. This is most likely due to the increase of cardiomyocytes and enhanced angiogenesis in the infarcted myocardium.
This study was supported by the Natural Science funds of Tianjin Province (10JCYBJC14000). The authors are very grateful for the sincere help and excellent technical support by the Key Laboratory of Artificial Cell, Institute of Hepatobiliary Disease of Tianjin Third Central Hospital.
Conflict of interest
The authors declare that no conflicts of interest exist.