Studies On MSCs Derived From HUCB Rrepairing Myocardium And Clinical Application Stem Cell Transplantation

Objectives1. Umbilical cord blood (UCB) as a source of hematopoietic stem cells has been successfully used in clinic for over 10 years. Could MSCs from UCB be isolated, cultured, amplified, and then induced into cardiomyocyte-like cells in vitro successfully? Using amplification and purification methods in vitro, we isolated and identified MSCs from UCB, and we investigated their biological properties after exposure to 5-azacytidine (5-AZA), a revulsant of promoting cardiomyocyte-like cell formation.2. We investigated whether cardiomyocytes resist to apoptosis caused by hypoxia after co-cultured with MSCs from UCB in vitro.3. MSCs from UCB were long-term cultured and amplified in vitro, their biological safety was observed after 5-AZA treatment.4. Granulocyte colony-stimulating factor (G-CSF) was used to mobilize bone marrow, after then, the mononuclear cells (MNCs) was isolated from peripheral blood to inject into the infarct site through cardiac catheterization. After treatment, the related indexes in those patients with acute MI were observed. Methods and ResultsMethods and resultsThe first part of the experiment:The normal fetal umbilical cord blood of healthy pregnant women was obtained. MNCs were isolated, and then cultured and The first part of the experiment:The normal fetal umbilical cord blood of healthy pregnant women was obtained. MNCs were isolated, and then cultured and passaged. Experiments were performed with cells from passage 3. Flow cytometry (FCM) was used to determine surface markers of cells (CD34, CD44, and CD90). After 10μmmol/L of 5-AZA treatment for 4 weeks, cardiomyocyte-like change was observed under inverted microscope. Transmission electron microscope (TEM) showed oval-shaped nucleus and bundles of filaments in the cytoplasm. Spindle dense bodies, abundant glycogen granules, large number of mitochondria and endoplasmic reticulum were found inside filaments. Cells with positive expression of GATA4 and cTnI were detected by immunofluorescence staining. Moreover, gene expression ofβ-myosin heavy chain (P-MyHC) was detected. After 5-AZZ treatment, cell growth cycle analysis and growth curve trace were performed.Results:The surface markers of MSCs from UCB after purification and amplification in vitro were in consistent with those of bone marrow counterpart. Cells had high purity from the 3rd generation. UCB-derived MSCs could differentiate into cardiomyocytes after 5-AZZ treatment in vitro, retaining their original characteristic with the inductive dose.The second part of the experiment:Human cardiac myocyte (HCM) ischemic injury model was established by exposure HCMs to hypoxia (95% N2-5% CO2). The results showed that hypoxia induced apoptosis of HCM in a time-dependent manner. Co-cultured with HCM in hypoxic conditions, UCB-derived MSCs could inhibit hypoxia-induced apoptosis.The third part of the experiment:Whether malignant transformation of UCB-derived MSCs occurred or not was determined by long-term cultivation and amplification in vitro, growth curve and growth cycle analysis after 5-AZA induction for 28 d, telomerase activity, G-banding patterns of chromosomal karyotpes, tumor formation, cell cycle-related gene and tumorigenicity. No abnormal chromosomal karyotpes were observed in UCB-derived MSCs after 5-AZA induction compared with that of un-induced MSCs. The fourth part of the experiment:Six patients with acute MI were selected in this study. One week before transplantation, G-CSF, a kind of stem cell mobilization reagent, was applied to mobilize bone marrow stem cells. MNCs were isolated and purified by cell isolation system, then were injected into the infarct site through cardiac catheterization. After stem cell transplantation, these patients were followed up for 1,3 or 6 months by ECG, left ventricular ejection fraction (LVEF) via cardiac ultrasound, and myocardial perfusion imaging (MPI). Compared with the control group, cardiac function in patients after stem cell transplantation improved significantly, and small R-waves appeared in infarct site determined by ECG, with more survival cells.Conclusions1. Umbilical cord blood is another source of MSCs other than bone marrow. UCB-derived MSCs provide a good cell donor for cardiomyocyte transplantation, which is another source of cardiac stem cells.2. Apoptosis of cardiomyocytes and hypoxia was time-related. Co-culture of human UCB-derived MSCs and HCM can resist against hypoxia-induced myocardial apoptosis effectively.3. The method of evaluating cardiomyocyte apoptosis at cellular level is established.4. UCB-derived MSCs in vitro have the similar growth rate with normal MSCs.5. No malignant transformation occurs in UCB-derived MSCs before and after 5-AZA treatment.6. The stem cell transplantation improves patients’ cardiac function significantly, and small R-waves are in infarct site on ECG, with more survival cells.