Inducing The Differentiation Of Human Parghenogenetic Embryonic Stem Cell Into Intestinal Epithelial Stem Cell

Background and Objective:Damage of intestinal epithelium including crohn’s disease, ulcerative colitis, short bowel syndrome, radiation enterocolitis, and so on, can cause malabsorption and even endanger the life of patient in severe case. Damage of intestinal epithelium is an intractable problem in clinic. At present, treating damage of intestinal epithelium with intestinal epithelial stem cell(IESC) which owns the potency of repairing the intestinal epithelium becomes a research focus at home and abroad. However, primary culture of IESC in vitro is difficult, which constraints the application of IESC in clinic. Human partheno genetic embryonic stem cell(hPESC) can avoid many problems such as transplant rejection and ethical issues. Therefore, hPESC is expected to become a significant cell source of IESC. In this study optimum culture condition of hPESC in vitro will be studied at first, then the feasibility and condition of inducing hPESC to differentiate into IESC in vitro will be explored.Methods:1. Trypsin digestion method was adopted to carry on primary culture of hFF. Then morphology of hFF was observed with inverted microscope, and hFF was identified by immunohistochemistry method.2.Three different culture systems of hPESC were established as follows:mouse embryonic fibroblast(MEF) as feeder-layer, human foreskin fibroblast(hFF) as feeder-layer and feeder-free system. Morphology of hPESC in three culture systems were observed respectively. Alkaline phosphatase activity and karyotypes analysis were used to detect the biological characteristics of hPESC. Culture condition of hPESC in vitro was optimized. 3. hPESC was induced to differentiate into definitive endoderm(DE) in vitro. Wnt3a and Activin A were adopted simultaneously as inducing factors to promote hPESC to differentiate into DE. Markers of DE including CXCR4、ECD、Sox-17and Gsc, were monitored dynamically with flow cytometry and real-time quantitative PCR, thus the time when the percentage of DE culminated was detected.4. Inducing DE to differentiate into IESC with inducing factor EGF in vitro. Markers of IESC including Msil and Hesl, were monitored dynamically with immunocytochemical double staining and real-time quantitative PCR, thus the differentiated time of IESC was detected in vitro.Results:1. hFF was obtained successfully by primary culture. Morphology and characteristic of hFF were verified with inverted microscope and immunocytochemical stain.2. Morphous of hPESC on hFF feeder-layer was regular and growth state of hPESC was well. hPESC on hFF feeder-layer was not apt to differentiate. After subcultured for25passages in vitro, hPESC still maintained biological characteristics and normal karyotype. Morphous of hPESC on MEF feeder-layer was regular and growth velocity of hPESC was stable. Self-differentiation of hPESC on MEF feeder-layer seldom occurred. After subcultured for15passages in vitro, hPESC still maintained biological characteristics. Morphous of hPESC in feeder-free system was regular and growth velocity of hPESC was slow. Self-differentiation ratio of hPESC in feeder-free system was extremely low. After subcultured for15passages in vitro, hPESC still maintained biological characteristics.3. After hPESC has been subcultured for15passages in vitro, morphous, adherence ratio, self-differentiate ratio and amplification velocity of hPESC, animal-derived contamination, workload and cost in three culture systems were considered thoroughly. We found that as feeder-layer of hPESC hFF owns a significant advantage. 4. Under the effect of Wnt3a and Activin A, hPESC was induced to differentiate into DE. The results of flow cytometry indicated that expression of CXCR4and ECD culminated on the second day. The results of real-time quantitative PCR displayed that expression of Sox-17and Gsc culminated on the second day. Therefore, the percentage of DE was highest on the second day.5. Inducing DE to differentiate into IESC with inducing factor EGF in vitro. Immunocytochemical double staining displayed that Msil+Hesl+cells culminated on the fifth day. The results of real-time quantitative PCR revealed that the expression of Msil and Hesl culminated synchronously on the fifth day. Therefore, the percentage of IESC was highest on the fifth day.Conclusions:1. hFF was obtained successfully by trypsin digestion method.2. Under the appropriate conditions all of three systems could sustain the growth of hPESC and keep hPESC undifferentiated.3. hPESC could keep biological characteristics and normal karyotype on hFF feeder-layer. As a kind of human-derived feeder-layer, hFF could sustain the growth of hPESC and keep hPESC undifferentiated. After various comparison, we found that hFF feeder-layer have a significant advantage.4. Combined application of inducing factors Activin A and Wnt3a can promote hPESC to differentiate into DE; the percentage of DE was highest on the second day. The hPESC induced with Wnt3a and Activin A on the second day is the ideal progenitor cell of IESC.5. Inducing factor EGF can promote DE to differentiate into IESC. The percentage of IESC was highest on the fifth day. It will provide potential cell source for the IESC transplantation treatment of damage of intestinal epithelium and theoretical basis for reconstruction of intestinal epithelium and intestinal tissue engineering research.6. hPESC was successfully induced to differentiate into IESC by a phased method.