Multipotential Differentiation Of BMDCs And The Mechanisms For Their Epithelial Differentiation

Stem cells are a special group of cells, which participate in the embryonic development and exist in various adult mature tissues. The most obvious characteristics of stem cells, which distinguish them from other mature cells, are their ability of self-renewing and multipotential differentiation. On account of their multipotential differentiation, they are believed to have a bright future in the regenerative medicine and tissue engineering. Based on their main origins, stem cells could be divided into two major groups:the embryonic stem cells (ESC), and adult stem cells (ASC). Theoretically, any type of tissues and cells could be derived from ESC. However, their wide use in tissue engineering filed has been limited by two problems:one is expensive cultivation costs, the other ethics debating. Accordingly, ASC have several advantages against ESC, which make them more interested for biological researchers, including their wide tissue origin, convenient isolation and expansion.Bone marrow is the major hematopoietic tissue in adult mammals. At the mean time it owns various stem cells, so it becomes the most important reservoir for adult stem cells. There are two popular groups of bone marrow stem cells which are investigated by scientists:one is hematopoietic stem cells (HSC), the other mesenchymal stem cells (MSC). In aspect of our current knowledge, the migration, proliferation and differentiation process of stem cells are the combination results of both cellular and humoral components in the microenvironment (also named niche) where they reside. The stem cell research is helpful for us to promote our understanding about various biological processes, including the individual development, tissue regeneration, and even tumorgenesis. In addition, therapeutic application of stem cells could promote wound healing and clinic management of kinds of human diseases by their wide differentiation spectrum and paracrine function.However, there are different opinions about the differentiation ability of bone marrow derived cells (BMDCs), and many problems need to be resolved by further study. Among all these problems, one is that many differentiation phenomena are induced by chemical reagents or recombinant inducing factors in vitro, so it remains unclear that whether these differentiation phenomena would occur in vivo. Therefore, it is of great importance to further study the differentiation potentiality and intrinsic mechanisms of BMDCs in vivo.The gastrointestinal (GI) tract is a critical organ, which is responsible for food digestion, nutrients absorption and immune response against both internal and external antigens. In accordance with these functions, the GI epithelium is one of the most vigorous tissues, which is completely renewed within 3-5 days. The rapid renewing is due to duplication and differentiation of intestinal stem cells (ISC). Then what would happen to BMDCs if they enter such an active microenvironment? Would they affect the epithelial function and GI motility? It has been reported that in both clinic patients and experimental animals donor's BMDCs could differentiate into GI epithelial cells after transplantation. This phenomenon provides us a new insight into differentiation lineage of BMDCs and a novel origin of GI epithelial cells. However, many key problems of the biological behavior of BMDCs in GI tract remain unclear, including migrating route, differentiation potentiality, mechanisms of epithelial differentiation and so on.In this study, whole body irradiation (WBI) was firstly employed to induce injuries in multi-tissues and multi-organs of C57BL/6 mice. Green fluorescent protein (GFP) transgenic mice were used as transplantation donors to establish bone marrow chimeric model between GFP mice and C57BL/6 mice. The migration, engraftment and differentiation of BMDCs were studied in main organs. At last we focused on the GI tract to investigate Cajal and epithelial differentiation of BMDCs. These data provided both theoretical guidelines and experiment supports for further study about BMDCs'differentiation spectrμm, epithelial differentiation mechanisms, and kinetics of epithelial cell renewing in GI tract.There were four maior parts in this dissertation listed as below:PartⅠ. Establishment and assessment of bone marrow chimeric modelIn order to study BMDCs efficiently, GFP expression level of BMDCs in GFP transgenic mice was primely confirmed. Then different doses of irradiation were used to destroy the hematopoiesis of C57BL/6 mice. Peripheral WBC counts at different points and the survival rate 14 days after experiment in different groups were compared. The level of GFP expression in bone marrow cells, HSC and MSC of long term chimeric mice had also been investigated.PartⅡ. Study of the multipotential differentiation of BMDCs in vivoThe repopulation effects of BMDCs on recipient's hematopoiesis of bone marrow and spleen were analyzed after transplantation. Then imunohistochemistry (IHC) and immune fluorescent (IF) staining were used to detect the engraftment and multipotential of BMDCs in different organs, including (1) mesoderm origin:bone marrow cells, spleen cells, and glomerular mesangium cells; (2) endoderm origin:pulmonary epithelial cells and hepatocytes; (3) ectoderm:cerebral neurons, skin hair follicle cells and epidermal cells.Part III. BMDCs repopulate irradiation injuried ICC in small intestineThe injury effects of irradiation on GI Cajal cells (ICC) were analyzed. Then BMDCs differentiation towards ICC and macrophages were investigated in GI muscularis.Part IV. Epithelial differentiation of BMDCs and the underlying mechanismsIntestinal epithelial differentiation of BMDCs was firstly confirmed. The level of SDF-1 expression in injuried intestine and its chemoattract influences on BMDCs were analyzed, and then the migration route of BMDCs in intestine was studied. How BMDCs differentiated into intestinal epithelial cells and what kind of stem cells in BMDCs had the capacity of epithelial differentiation were further investigated.Following main results and conclusions were obtained:1. Combination of lOGy WBI and BMDCs transplantation could establish stable chimerism between GFP mice and C57BL/6 mice.Using lOGy irradiation and transplantation of 1.5x107 GFP BMDCs, the survival rate came to more than 93% at 14 days after transplantation, and the peripheral WBC count recovered near to the normal condition. Chimeric mice could survive long time with hair graying. GFP+ BM cells were more than 90% even 14 months after BMT, among which HSC and MSC were positive at 99.95%and 96.84%respectively. This stable long term chimerism provided us an ideal model to explore the differentiation spectrum of BMDCs.2. Systemic observation of BMDCs differentiation was done in vivo for the first time10Gy WBI caused systemic damages, and BMDCs participated the tissue repairing program in both irradiation sensitive and insensitive organs. The early engraftment of BMDCs in different organs depended on the radiation sensitivity and injury degree. In the early stage BMDCs preferred to migrate into BM and spleen to reconstitute recipient's hematopoiesis. With hematopoiesis recovery the number of BMDCs increased in other organs. It was believed that increased BMDCs in organs were from early engrafted BMDCs after transplantation. BMDCs engrafted in different organs had a wide differentiation capacity in vivo, including BM cells, spleen cells, glomerular mesangium cells, pulmonary epithelial cells, hepatocytes and oval cells, cerebral neurons, skin hair follicle cells and epidermal cells. The results indicated that BMDCs had the capacity to differentiate into non-mesodermal originating cells, in other words, they had the transdifferentiation ability.3. Irradiation caused damage to Interstitial cells of Cajal (ICC)ICC generated slow wave electricity and were responsible for GI motility. Radiation not only damaged GI epithelium and lamina propria, but also caused damage effects on ICC, including reduced cell count and morphological changes to ICC, in addition, especially reduction of functional molecule c-Kit. The decrease of cell count had a certain linear relationship with radiation dose. ICC density 3days after radiation reduced to 53.4% as to normal status, and c-Kit 74.5%. It was the first report of radiation caused ICC damage, and this explained why patients and animals suffered from intestinal obstruction and invagination after radiation.4. BMDCs were found to be the origin of GI ICC for the first timeICC was sensitive to many harmful factors, so it was important to study the replacement origin of ICC after injury. It was demonstrated that ICC did not originate from neural ectoderm, but from the mesenhyme. Recently, it was shown that in neonate stomach ICC arised from a special type of muscular cells. BMDCs could differentiate into GFP and c-Kit double positive ICC in chimeric intestinal walls, and they ranged from 0% to 11.2%. It was the first report concerning extraintestinal origin of ICC.5. BMDCs could spontaneously and continuously form donor derived IECBMDCs in recipient's intestinal epithelium expressed epithelial marker pan-cytokeratin (PCK), but did not express leukocyte marker CD45, indicating BMDCs could form intestinal epithelial cells. Some BMDCs in epithelium could also express Chromogranin A, suggesting they were enteroendocrine cells. Under immune electron microscope it was found that donor derived epithelial cells got the correct ultrastructural characteristics. IF image analysis and flow cytometry (FCM) evidenced that the percentage of donor derive IEC took a percentage of 9.41%-16.07% in all the IEC.6. Radiation damaged intestine attracted BMDCs to migrate towards intestineSDF-1/CXCR4 axis was the most important chemoattract axis. The mRNA level of SDF-1 was elevated at 6h after irradiation, and at 24h it increased 6 folds. The increased SDF-1 protein sustained 8d after radiation. These indicated that radiation damaged intestine reconstituted epithelium depending on not only ISC, but also BMDCs. MSC expressed CXCR4, the receptor for SDF-1. And they could respond to the increased SDF-1. IEC-6 at 24h after lOGy irradiation could attract BMDCs significantly, and AMD3100, the SDF-1 antagonist, could block the chemoattract effects. After 3h reaction, MSC increased 43.8% more than control group, and HSC increased only 15.1%more than control. This indicated that damaged intestinal epithelial cells (IEC) had more mobilizing effects on MSC than HSC, so we needed to spend more efforts on MSC. During early stage, BMDCs engrafted into bottom of lamina propria, then migrated towards tip of lamina propria, and no BMDCs would enter epithelium. At 1 month after transplantation BMDCs fulfilled the lamina propria and appeared to have the ability to form donor derived IEC.7. Cell fusion was not the only possible mechanism for donor derived IECIn female GFP mice to male C57 mice BMT model, GFP expression was used as donor marker, and Y-FISH as recipient marker. On serial sections, coexpression of GFP and Y-FISH was analyzed. Taking both the efficiency of GFP IHC and Y-FISH into account, it was fond that the double positive IEC only took a proportion of 53.5%among all the donor derived IEC, and the GFP single positive rate was 46.4%. There was no significant difference between these two groups, indicating cell fusion was not the only reason for donor derived IEC.8. MET was another possible pattern for the formation of donor derived IECIn chimeric intestine donor derived IEC were mainly distributed at the villous part of the villi-crypt unit. The distribution of these cells was in a separate and discontinous pattern, and few positive cells could be detected in the cryptal epithelium. Ki67 positive BMDCs could migrate into villous epitheliμm, and these round-shaped cells located at the base of IEC. There were also some Ki67 positive BMDCs in the surface of epitheliμm. In the villous epitheliμm 23.6%BMDCs were positive for Ki67, and there were even Ki67 positive donor derived epithelial cells. The results supported the hypothesis that proliferative BMDCs could migrate from the lamina propria to the epithelium, and be induced to gain morphological features and phenotype of IEC in an epithelial microenvironment.9. MSC had a priority to be induced to IEC phenotype than HSCWhen MSC and HSC were cocultured with IEC-6, an intestinal epithelial cell lineage, MSC could be detected to have the expression of E-Cadherin at 24h, and PCK at 48h. However, HSC could be detected the expression of E-Cadherin and PCK at 7d. Such a result indicated that MSC were more ready to form IEC than HSC.10. MET might be the mechanism of IEC phenotype formation of MSCBM was harvested and cultured from abortion fetus with permission. MSC at P10 had the satisfied morphological appearance, surface markers, cell cycle data, and multipotential differentiation ability. When hMSC were cocultured with IEC, they were found to be more proliferative than normal control. The normal cultured hMSC did not express E-Cadherin by PCR analysis and IF staining. E-Cadherin expression increased 6.46 folds after 3d coculture and 7.66 folds after 7d coculture. The intestinal specific cytokeratin Krt20 increased 3.77 folds at 3rd day and 6.38 folds at 7th day. The data indicated that in the coculture system increased E-Cadherin expression in hMSC might start MET process and then drive Krt20 expression, so hMSC might obtain the IEC phenotype by a MET mechanism.