Study On Evidence Of Origin Of Adult Stem Cell

Background and Purpose This thesis aims at testing the hypothesis that bonemarrow is the source of adult tissue stem cells. In the past few years, the plasticity of adult stem cells (ATSCs) has become the hot point in biomedical research. However, it raises doubt now. A lot of negative experimental results were published in succession, even in Nature and Science. The reason that the remarkable plasticity of adult stem cells has drawn so much attention is that not only it is regarded as a significant breakthrough in developmental biology but also offers the potential for medical use of ATSCs for the reconstitution of injured tissues in trauma and diseases. The key issue about plasticity is whether it is natural or just artificial like Dolly, the cloned sheep. Two important aspects remain to be clarified about the query, one is the relationship between various adult tissue stem cells, or their real identity and origin, the other is whether they are capable of circulating in the blood. It has been well known that the fates of stem cells largely depend on their tissue environment. Transdifferentiation will not occur until tissue stem cells leave their original tissue environment and migrate to a new location. Most of the reports about the plasticity of ATSCs were the results of in vivo experiments in which changes of their environment were man-made. The success of cloned animals like Dolly indicated that even specialized somatic cells could also be reprogrammed to the primitive condition and then follow another development from the beginning like zygote. But obviously, this won't occur naturally. Bone marrow derived stem cells are among the most widely studied ATSCs for plasticity, especially hematopoietic stem cells (HSCs) and mesenchymal stem cell(MSCs). HSCs can differentiate into blood cells and immune cells and circulate in peripheral blood. Recent findings suggest that HSCs have the potential to differentiate into non-hematopoietic tissue cells, such as muscle fibers, hepatocytes, microglia astroglia and neuronal tissue.Whether MSCs circulate in the blood is inconclusive, but they exhibit remarkable plasticity both in vitro and in vivo, with the potential to differentiate into all three germ layer derived differentiated cells, including neuron, muscle, kidney and lung, etc. Besides, it has long been known that bone marrow derived cells play a critical role in wound healing as wounds enroll many mature inflammatory cells which provide cytokines that orchestrate the healing process. Thus the hypothesis is put forward, and will be tested in many aspects.MethodsExperiments were designed to characterize the role of bone marrow stem cells (BMSCs) in tissue physiological renewal and pathological repair of three germ layers derived organs and the circulation and distribution of BMSCs in adult tissues under natural condition. Then, the effects of transplantation of BMSCs on diabetes and mobilization of autologous BMSCs by G-CSF on severe acute pancreatitis(SAP) were investigated.Skin, heart and pancreas were selected as examples for ectoderm, mesoderm and endoderm derived tissues, respectively. Each tissue had normal and injured groups. New Zealand rabbits were used as experimental animals. Their bone marrow were aspirated from iliac crest, and monocyte of bone marrow (MBM) were fractionated by density gradient centrifugation, then MBM were divided into two groups of stem cells, one group was HSCs which suspend in media while cultured in vitro, the other was MSCs which are adherent to plastic dishes. The three groups of BMSCs, MBM, HSCs and MSCs, were stained with Hoechst33258, a fluorochrome, which is capable of binding with nuclei. The nuclei labeling was fulfilled by culturing BMSCs together with the dye for 15min in vitro, then washing thoroughly with PBS to remove the extra free dye. The labeled BMSCs were then transplanted into their original medullary cavity. Three days later, three kinds of tissue injured models were induced.Excisional wounds of skin were made by lifting the skin with forceps andremoving a full thickness portion of skin (to the subcutaneous fat) using a curved scissors. Heart injury was induced by ip 5mg/kg of isopenaline(ISO). Pancreatic injury was induced by ip 1 mg/kg of L-arginine. Two weeks after the injury was induced, all the rabbits were sacrificed, and were intracardial perfusion of cold phosphate-buffered salt solution(PBS) to avoid blood cell contamination.Then wounded and normal tissues were harvested. Samples were snap-frozen and sectioned on a cryostat. The presence of labeled BMSCs in the cryostat prepared sections was examined directly by fluorescence microscopy. The positive sections were chosen for further immunofluorescence assay. Anti- Pan cytokeratin immunofluorescence staining was performed in skin section to determine whether the incorporated BMSCs differentiated into epidermal cells. Anti- a -actin immunofluorescence staining was performed in heart section to determine whether the incorporated BMSCs differentiated into myocardial cell. And anti-CK19, anti-insulin and anti- glucagon immunofluorescence staining were performed in pancreatic section to determine whether the incorporated BMSCs differentiate into mature pancreatic cells.The distribution of BMSCs in adult tissues. The mice from same mother were used as experimental animals, the males were taken as donor of BMSCs, and the females as recipient. Bone marrow was obtained by flushing the femurs of male dornor mice, and MBM was isolated by density gradient centrifugation, then HSCs and MSCs were separated as described above. Three groups of BMSCs, MBM, HSCs and MSCs, were then transplanted to female recipient via tail veins injection at a dose of 10' cells per mouse. Two weeks later, recipient mice were sacrificed. They were given a perfusion of 20 mL cold PBS at a flow rate of 4 mL/minute. Perfusate was pumped into the left ventricle of the heart and removed by means of the right atrium. And then 11 organs were taken out, including heart, liver, spleen, lung, kidney, brain, smal intestine, stomach, pancreas, skin and muscle. Tissue DNA was extracted by phenol-chloroform extraction, and PCR was performed to determine the presence of SRY region of Y chromosome from male donor in above tissues.The effect of transplantation of BMSCs on diabetes induced by alloxan in mice. Diabetes was induced by ip 200mg/kg of alloxan in female mice. Male micewere used as BMSCs donors. The bone marrow were obtained from femurs and tibias and fractionated by density gradient centrifugation and MBM were collected from the interface. MBM from male donors were transplanted to female via tail vein injection at a dose of 107 cells per mouse. 7, 20 and 50 day after MBM transplantation, female mice were sacrificed. Blood and urine glucose level were determined, HE staining was performed in pancreas, and the presence of Y chromosome in liver, bone marrow, spleen and pancreas was determined by PCR.Protective effect of autologous BMSCs mobilized by G-CSF in SAP mice. SAP was induced by ip 2g/kg of L-arginine in females as described above. Mice were divided into four groups, G-CSF pre-treatment, BMSCs transplantation, SAP, and normal control group. The death rate within 72h post induction of SAP, serum amylase level and pancreatic pathologic injury were examined. Meanwhile, the presence of Y chromosome from male donors in female pancreas was determined by PCR.ResultsAll three groups of BMSCs participate in repopulation of skin, heart andpancreas. They were all present in both normal and wounded dermis, scaterred innormal tissue, but clustered in wounded areas. BMSCs present in normal skindemonstrated by negative staining for anti-pan cytokeratin, while there were somepositive cells for this immunofluorescence assay in injured dermis, which indicatedthat they might transdifferentiate into epidermal cells. No BMSCs were present inintact hearts, but they all appeared in injured cardiac tissues without positive cells foranti- a -actin immunofluorescence staining. All three groups of BMSCs, MBM, HSCsand MSCs, were present in both normal and injured pancreas. In normal pancreas, nopositive cells for three kinds of immunofluorescence staining were found forincorporated BMSCs. But in injured pancreas, there were some positive cells foranti-CK19 staining, but no positive cells for anti-insulin and anti-glucagon staining,which suggested that some of BMSCs might differentiate into pancreatic stem cells,but none of them became functional cells of islets.Y chromosome was present in all 11 kinds of tissues as shown by PCR, which indicated that BMSCs might be capable of distributing to almost all the adult tissues and provide prerequisite for differentiation into corresponding tissues.Transplantation of allogenic BMSCs reversed hyperglycemia and high level of urine glucose in diabetic mice induced by alloxan. Pancreatic pathologic damage improved significantly in comparison with that of diabetic control group. The presence of Y chromosome in damaged pancreas indicated that transplanted BMSCs had incorporated into pancreas.Mobilized autologous BMSCs protected pancreas from severe damage during SAP. The results showed that both G-CSF pretreatment and BMSCs transplantation group had reduced death rate within 72h compared with that of SAP control group. The death rate in these three groups was 8%, 10% and 34%, respectively. The former two groups also had reduced level of serum amylase in comparison with SAP group. In consistency with these observations, both gross and microscopic examinations revealed that the pathological changes of SAP in animals pretreated with BMSCs transplantation or G-CSF injection were considerably attenuated as compared with that in controls. Y chromosome appeared in damaged pancreas in BMSCs transplantation group, which indicated that incorporation of BMSCs in injured tissues might be the mechanism of protective effect of mobilized autologous BMSCs on pancreas during SAP.ConclusionsAll the four parts of this thesis support the hypothesis that bone marrow is the source of adult tissue stem cells. First, under natural conditions, BMSCs participate in the renewal and repair of tissues derived from three germ layers. Second, BMSCs circulate in blood normally, which provides extensive prerequisite for bone marrow act as the source of adult tissue stem cells. Third, both BMSCs transplantation and autologous mobilization are effective in curing pancreatic disorders, which indicated...