Intravenous Transplantation Of Adipose-derived Mesenchymal Stem Cells Results In Functional Recovery After Traumatic Brain Injury In Rats

BackgroundRecently, Lin Yuan has put forward a new hypothesis, fasciaology. In fasciaology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of undifferentiated cells of unspecialized connective tissues. The other is the functional system, which is consisted of differentiated functional cells and is enclosed by the supporting-storing system. The undifferentiated stem cells in the supporting-storing system incessantly differentiate into functional cells. The supporting-storing system throughout the body regulates the functional and living status of differentiated cells and provides a stable internal environment for the survival of these cells. Adipose tissue is a an important part of the supporting-storing system, and adipose-derived mesenchymal stem cells (ADMSCs) are main stem cells reserved in this system. We investigated the difference of stem cells reserved in different site, for example, in greater omentums and subcutaneous adipose tissues, by comparing the characteristics of multilineage differentiation potential and cell surface markers of ADMSCs. This research will contribute to fasciaology and cell therapy. In recent years, interest has rapid grown in the research of ADMSCs. Being one kind of mesenchymal stem cells, ADMSCs have the capacity to selfrenew for indefinite periods and can differentiate to many different cell types. Mesenchymal stem cells can be isolated from several organs, such as bone marrow, fat, umbilical cord blood, peripheral blood and skeletal muscles. A significant number of investigations have focused on mesenchymal stem cells derived from bone marrow, which can differentiate to multiple cell phenotype, including bone, fat, cartilage, skeletal muscle and neural progenitor, under appropriate culturing conditions. Bone marrow mesenchymal stem cells were first identified and are one of the most widely used stem cell sources. Therapeutic potential of transplantation of them is invigorating. Compared with bone marrow mesenchymal stem cells, ADMSCs do have an equal potential to differentiate into multiple cell phenotype. However, the simple surgical procedure, the easy and repeatable access to the subcutaneous adipose tissue, and the uncomplicated enzyme-based isolation procedures make ADMSCs most attractive for researchers. Being a new source of therapeutic stem cells, ADMSCs should be given more attention to.Autologous ADMSCs as seed cells are ideal for autologous stem cell transplantation. In order to obtain a large number of autologous seeded cells, ADMSCs should be idolated from adipose tissue and cultured in vitro. As the ADMSCs donor, organisms are often in pathological situation (such as trauma). It should be investigate that whether there are sufficient ADMSCs in such situation and the influence of trauma on the multiple differentiations and cell surface markers.Traumatic brain injury (TBI) is a major health problem in the world. The therapy of TBI faces many difficulties as a result of the limited ability of the central nervous system for self-renewal and complicated pathological processes present in the state of the brain disorder. Currently, there is no effective clinical treatment to promote recovery after TBI. However, a new interventions, cellular therapy, is being explored in experimental studies. Considerable hope is vested in cellular therapy, in which transplanted cells would be the source neurotrophic factors and could also be the source of new cells. We investigated if intravenous transplatation of ADMSCs results in functional recovery after TBI in rats and the influence of cell transplatation on brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) release.Objective1. To isolate and cultivate ADMSCs from SD rat and conduct immunophenotypic characterization with the following cell surface markers:CDllb, CD29, CD45, CD49d, CD90 and CD 106. And to induce adipogenic and osteogenic differentiation of ADMSCs and conform their differentiation potential.2. To investigate the difference of the immunophenotypic characterization by comparing the cell surface markers profile and the proliferative capacity of ADMSCs reserved in viscera (greater omentums) and subcutaneous adipose tissues in Sprague-Dawley rats. And to compare the quantitative osteogenesis assay and the quantitative adipogenesis assay.3. To investigate the influence of skin wound on the yield, growth characteristics and cell surface markers of ADMSCs in rats.4. To investigate the effect of intravenous transplatation of ADMSCs on functional recovery after TBI in rats, the migration of ADMSCs in the injured brain and the influence of cell transplantation on BDNF and GDNF.Methods1. The isolation and culture of ADMSCs of SD rats, multi potentiality and the characterization of cell surface markers: ADMSCs of 6 Sprague-Dawley rats from inguinal fat pads were isolated and cultured. Briefly, the adipose tissues were mechanically dissociated and digested with collagenase type I. The adipocytes were separated from the stromal vascular fraction by centrifuging the suspension. The cells in the stromal vascular fraction were plated in flasks at a density of 8000-10,000 cells/cm2. The cells were cultured with Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. After 24 hours, the non-adherent cells were removed and the adherent cells were expanded by serial passage. The morphological characterization of ADMSCs was observed using phase-contrast microscopy.ADMSCs at passage 4 were cultured under adipogenic and osteogenic condition to conform their differentiation potential. The morphological characterization of inductive cells was observed using histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation.The immunophenotypic marker including CD11b, CD29, CD45, CD49d, CD90 and CD106 of ADMSCs grown for 4 passages was determined using flow cytometry.2. The comparison of ADMSCs from viscera (greater omentum) and subcutane ous:ADMSCs were isolated and cultured from greater omentums and inguinal fat pads of 6 Sprague-Dawley rats. The morphological characterization of ADMSCs was observed using phase-contrast microscopy. ADMSCs at passage 4 were cultured under adipogenic and osteogenic condition to conform their differentiation potential. The morphological characterization of inductive cells was observed using histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation. ADMSCs from two sites were plated in 6-well plates. Adipogenic differentiation was induced by culturing ADMSCs in adipogenic medium and assessed using an Oil Red O stain as an indicator of intracellular lipid accumulation. For the quantitative adipogenesis assay the cells were de-stained with isopropyl alchohol. The amount of Oil Red O was determined by measuring the optical density of the solution at 510 nm. Osteogenesis differentiation was induced by culturing ADMSCs in osteogenesis medium and examined for extracellular matrix calcification by Alizarin Red staining. For the quantitative osteogenesis assay, the cells wer de-stained with 10% cetylpyridinium chlorid. The amount of Alizarin Red was determined by measuring the optical density of the solution at 560 nm. The average optical density of wells represents the quantitation of the levels of differentiation in ADMSCs.The ADMSCs at passage 4 from two sites were seeded in 24-well plates. Every 24 hours after seeding, cells from 3 wells were harvested and cells in each well were counted twice. Average ADMSCs numbers were plotted against the number of days cultured. The growth curves were investigated and the logarithmic growing phase of the cells was determined. The population doubling time was calculated using the formula:(days in logarithmic phase)/((logN2—logNl)/log2), where N1 is the number of cells at the beginning of the logarithmic growing phase and N2 the number of cells at the end of the logarithmic growing phase.The phenotypical marker profile including CD11b, CD45 and CD90 of ADMSCs from two sites grown for 4 passages was determined using flow cytometry.3. The influence of skin wound on adipose-derived mesenchymal stem cells harvested from subcutaneous adipose tissue in rats:6 Sprague-Dawley rats were suffered skin incision in left inguen and the wounds were sutured. After 7 days, ADMSCs of injured rats and 6 normal rats from right inguinal fat pads were isolated and cultured. The yield of stromal vascular fraction cells and adherent cells were investigated. Both kind of ADMSCs at passage 4 were induced to adipogenic and osteogenic differentiation. Growth kinetics were investigated and doubling times were determined. The phenotypical marker profile including CD11b, CD29, CD45, CD49d, CD90 and CD106 of ADMSCs grown for 4 passages was determined using flow cytometry.4. The influence of intravenous transplantation of ADMSCs on functional recovery after traumatic brain injury in rats:Traumatic brain injury model:Experimental TBI was induced in rats by a cryogenic injury model.36 SD rats (weighting 200 to 220 g) were randomly divided into three groups, the experimental group (n=12), the control group (n=12) and the normal group (n=12). The experimental group and control group animals were anesthetized with chloral hydrate intraperitoneally. Under surgical microscope the skull was exposed by a midline incision and a circular hole (approximately 5 mm in diameter) was drilled on the skull over the left temporoparietal cortex leaving the dura intact by using a dental drill. The precooled copper probe (-80℃) 4 mm in diameter with 10-g weight was lowered onto the dural surface and kept in place for 60 s. The skin was sutured. The rats in normal group were not injured.Intravenous transplantation of ADMSCs:ADMSCs of SD rat were isolated and cultured. ADMSCs at passage 4 were harvested after proliferating in the medium added 5-bromo-2-deoxyuridine (BrdU) (10μmol/L). One day after TBI, the rats in the experimental group were injected with ADMSCs labled with BrdU via the tail vein(3×106 cells in 1ml saline/each).Determination of cognitive function:Morris water maze tests were performed from day 11 to 14 after TBI between 9:00 and 17:00. The time to reach the platform (latency) was measured. The day after the acquisition phase (day 4), a test was conducted by moving and revealing the platform. The time reached the target, the visible platform, from the opposite quadrant was recorded.Immunofluorescent staining:Three rats in every group were anesthetized after Morris water maze tests. They next received intracardiac perfusion of 4% paraformaldehyde in PBS. Rat brains were postfixed overnight and then routine paraffin sections were made. Serial transverse sections at a thickness of 5μm were cut through the injured district. The expressions of the incorporation of BrdU were investigated using immunofluorescent staining and the migration of transplanted ADMSCs were determined.Quantitative analysis of BDNF and GDNF:The other 9 rats in each group were sacrificed by rapid decapitation on day 15. The brains were quickly removed and stored in liquid nitrogen until use. Western blott was used for measuring levels of BDNF and GDNF. The images were captured by Kodak Image Station 2000 MM system and data were processed using image processing software Image Tool 3.0. The levels of BDNF and GDNF were expressed as the ratio of studied protein versus actin.Results1. Primary culture cells showed fibroblastic-like morphologic characteristics. Within one to four passages, ADMSCs appeared as a monolayer of large, flat cells, and became relatively homogeneous in appearance as the cells were passaged. ADMSCs had the ability to differentiate along adipogenic and osteogenic lineages. When cultured under adipogenic condition for 2 weeks they were induced toward the adipogenic lineage. A fraction of the cells contained multiple, intracellular lipid-filled droplets that accumulated Oil Red-O. The Oil Red O-containing ADMSCs exhibited an expanded morphology with the majority of the intracellular volume occupied by lipid droplets, consistent with the phenotype of mature adipocytes. When cultured ADMSCs were exposed to an osteogenic induction medium, they aggregated and formed calcium deposits after 2 weeks. An alizarin red stain for precipitated calcium salt was performed on differentiated cells. Calcification appears as red regions within the cell monolayer. The immunophenotypes analysis revealed that the rat ADMSCs were positive for CD29 and CD90, but negative for CDllb, CD45, CD49d and CD 106.2. The morphological characterization of ADMSCs harvested from greater omentums and inguinal fat pads were almost the same. Primary culture cells and cells within one to four passages from two sites showed the same fibroblastic-like morphologic characteristics. Both of them have the capacity to differentiate toward the adipogenic and osteogenic lineages. The average optical density representing the quantitation of the levels of differentiation was confirmed after ADMSCs were induced in adipogenic and osteogenesis differentiations. The abilities to differentiate along adipogenic and osteogenic lineages showed no significant difference between two kinds of cells. The growth curves of cultured ADMSCs at passage 4 from the two sites showed no obvious difference. The population doubling times based on the growth curves were similar between ADMSCs from greater omentums and from inguinal fat pads, respectively being 0.72±0.05 days and 0.73±0.06 days (P> 0.05). The frequencies of ADMSCs exhibiting CD11b,CD29,CD106 and CD90 phenotype are similar (P> 0.05). The frequencies of ADMSCs exhibiting CD45 and CD49d is different (P<0.05).3. The morphological characterization of these ADMSCs were almost the same in two groups. The yield of stromal vascular fraction cells and adherent cells from injured rats (respectively being 0.47±0.20×104/mg and 1.29±0.52×104/mg) were fewer than them from normal rats (respectively being.1.43±0.17×104/mg and 3.84±0.54×104/mg) (P<0.05). ADMSCs from injured and normal rats could differentiate into adipogenic and osteogenic lineages. The growth curves of these two kinds ADMSCs showed no obvious difference. The population doubling times based on the growth curves were similar, respectively being 1.11±0.06 days and 1.09±0.05 days (P> 0.05). The immunophenotypes analysis revealed that ADMSCs from two groups were the same positive for CD29 and CD90, but negative for CD11b, CD45, CD49d and CD106. The frequency of CD11b+,CD29+,CD45+,CD49d+,CD90+and CD106+ ADMSCs derived from injured rats and normal rats are similar (P>0.05).4. Grafted ADMSCs improved cognitive function in rats after TBI.Morris water maze tests:All the rats were tested, not having obvious health problems such as weight lost. There was an overall significant effect of treatment on the escape latency throughout the behavioural experiment of the subjects (P<0.05). The escape latencies to platform in experimental group were significantly shorter than those in control group on days 1, 2,3 and 4. The escape latency was no significant difference among the three groups on the fifth day.The migration of transplanted ADMSCs was determined by immunofluorescent staining of BrdU. In animals of experiment group we examined, many of the grafted ADMSCs were found in the injured cortex and were not found in the normal cortex and other districts.The levels of BDNF and GDNF were significantly higher in experimental group than them in control group (P=0.000).Conclusions1. The ADMSCs harvested from SD rat obtained through an enzyme-based isolation procedures have the ability to differentiate along adipogenic and osteogenic lineages.. The results revealed the immunophenotypic characterization of them is consistent with mesenchymal stem cells. 2. The immunophenotypic characterizations of ADMSCs reserved in greater omentums and subcutaneous adipose tissues are not absolutely similar. They all have the ability to differentiate along adipogenic and osteogenic lineages. The quantitative osteogenesis assay and the quantitative adipogenesis assay are the same.3. The yield of ADMSCs in injured rats is decreased and influenced by skin wound. ADMSCs cultured from injured and normal rats are similar in multiple differentiations, growth characteristics and cell surface markers.4. Intravenous transplatation of ADMSCs results in functional recovery after TBI in rats. The grafted ADMSCs can migrate to the injured cortex. The cell transplantation results in a higher level of BDNF and GDNF in the injured brain.