The Immunobiological Development Of Bone Marrow Mesenchymal Stem Cells In The Course Of Neuroal Differentiation

It is known to all that the death rate and disability rate of centralnervous system(CNS)lesion are high. Although, much progression wasmaken in drug treatment and surgery treatment in the last century, thosetreatments can not fundamentally cure the impaired nerve cells. Thus it isof great importance to search for an effective method.Now cellular transplantation has become one of the most potentialmethods. Bone marrow mesenchymal stem cells (MSCs) are a kind ofadult stem cells. Compared with the stem cells resource, MSCs are mucheasy to be obtain from bone marrow. Additionally, they are expanded invitro by using simple procedures, and can be induced into the cells ofthree germ layers. Under certain conditions, MSCs can differentiate alongneuroal lineage, which offer exciting possibilities for nerve injury therapy.After human bone marrow MSCs were injected in the striatum of ratsfor 5～27 days, approximately twenty percent of transplanted cells werelive and migrated on the way of nerve stem cells migration (Aziz, 1998).After injected in lateral cerebral ventricle of neonatal mice for 12 days,bone marrow MSCs migrated and concentrated in neuron district.Neurofilament(NF) positive cells were found in the brainstem formatioreticularis, Which indicated that MSCs might differentiate into neurons.In the striatum and stratum moleculare hippocampi, glial fibrillary acidicprotein (GFAP) were expressed in MSCs, Which demonstrated thatMSCs might differentiate toward astrocytes too (Kopen, 1999).It might be one of the best choices that autogeneic MSCs wereused to therapy nervous system diseases. However, the donators arelimited. Therefore, the method of allogenic transplantation was attempted.Previous studies reported that undifferentiated MSCs expressedintermediate levels of human leukocyte antigen (HLA) classⅠmolecules, without HLA classⅡmolecules, or the co-stimulatory molecules. It wasreported that both baboon and human MSCs escaped alloreactive T-cellrecognition in mixed lymphocyte cultures (MLC) (Bartholomew, 2002).Transplantation of allogeneic major histocompatibility (MHC)mismatched MSCs into baboons was well tolerated(Bartholomew, 2002).When co-transplanted with hematopoietic cells, MSCs might decrease therate of acute and chronic graft-versus-host disease (Guo, 2000; Kim,2002). MSCs which differentiated along osteogenic, chondrocytal andadipocytal lineage retained their immunoprivilege andimmunosuppression in vitro (Le Blanc, 2003; Liu, 2006). These dataindicate that MSCs is one of ideal organize engineering cells.However, different opinion was proposed in the last year. WhenMSCs were transplanted into allogenic mice, MSCs were unable tolong-term surviving and CDS+ cells and natural killer cells wereincreased. (Eliopoulos, 2005).After MSCs from donator were implantedinto receptor, immune reaction lied in between MSCs and memory T cells(Nauta, 2006).With MSCs differentiating along osteogenic lineage, theirimmunosuppression in new Zealand albino rabbit was significantlydecreased(Liu, 2006). Datas from above indicated that MSCs mightarouse immunological rejection in vivo, and could not been transplantedin MHC mismatched individuality.CNS has been thought to be an immune privileged organ (Billingham,1953; Barker, 1977). However, some investigations recently dedicatedthat it was not absolute and the privileged status of the brain was regardedas the result of a balance between immune privilege and effectiveresponses (Brabb,2000).There was a potential threat of graft rejection dueto MHC incompatibility. Current studies of transplantation in brainmainly focued on the morphological characteristics and function ofneuroal differentiated MSCs. Substitution is one of mechanisms in neural transplantation with MSCs. However, the immunological evidence ofMSCs differentiated along neuroal lineage has not been identified untilnow. We don't know whether these neuroal differentiated cells will belong-term surviving in brain.Neonatal hypoxic-ischemic brain damage(HIBD) is a brain injuredisease due to asphyxia during foetus or neonatal period. In spite of thefast development in perinatology, HIBD remains one of main reasonswhich cause neonatal death, cerebral palsy, mental retardation andepilepsy (Spong, 2005). MSCs transplantation gives a new way in HIBDtherapy. It is unclear whether immunological rejection will be induced fordestroying the kinetic balance immunologic of brain.In this paper, we intend to observe the immunogenicity of MSCsdifferentiating along neuroal lineage and the effect on the immunereaction after transplantation in the brain of neonatal HIBD model.Although the immunobiology of MSCs in future cell therapy remains tobe tested and validated, our study is increasingly available for thispurpose, and will provide the possible of more effective therapies forneural disorders.PartⅠ: The immunogenicity of human bone marrow mesenchymalstem cells in the course of neuroal differentiationObjective: To investigate the immunophenotype of bone marrowMSCs differentiating along neuronal lineage and the effect on theproliferation of allogenic T cells.Methods: Human MSCs were isolated and expanded from bonemarrow. For neuroal differentiation, MSCs were cultured in a serum-freemedium for one week or two weeks. The serum-free medium weresupplemented with basic fibroblast growth factor (bFGF) and epidermalgrowth factor (EGF). In order to confirm whether the differentiated cells are in the neuronal lineages, the cells were identified by the methods ofimmunofluorscence and western blotting. According to the period ofMSCs differentiating along neural cells, the cells were divided into 3groups: undifferentiated MSCs group (group MSCs), differentiated forone week group(group Neu1) and differentiated for two weeksgroup(group Neu2). The immunophenotype of cells were analyzed byflow cytometry and western blotting analysis. Cells of each group werecultured with allogenimic T lymphocytes and T lymphocytes proliferationwas assessed by [~3H]-thymidine incorporation using a liquid scintillationcounter. In some experiments, IFN-γ100 U/ml was added to the culturemedium for 2 days prior to analysis. Spss11.0 package and Excel7.0software were used in statistical analysis.Results: MSCs from human bone marrow were successfullycultured in our study. At the passage one, the cells were heterogeneous.At passage four, cells were homogeneous. Inverted phase contrastmicroscope demonstrated that the cells had a spindle-shaped andfibroblastic appearance. Furthermore, flow cytomtry indicated that theMSCs at this period exhibited the positive labeling with CD29, CD105,CD44, but negative staining with CD34, CD14, and CD45. This datademonstrated that the cultured cells expressed surface markers of MSCs,and did not express surface markers of haemopoietic stem cells or surfacemarkers of endotheliocytes. For neuroal differentiation, MSCs werecultured in a neuroal medium for one week or two weeks. After 5～7 daysof culture in neuronal medium, the cytoplasm in the MSCs retractedtowards the nucleus; after 2 weeks of treatment, many cells presentedwith neuronal morphology including: a small cell body and longextensions. Indirect immunofluorescence analysis demonstrated thatsome of untreated MSCs expressed nestin, but NF-L and neuron specificenolase(NSE) were not expressed. After MSCs were cultured in neuroal medium for two weeks, the number of nestin positive cells increased andexpressed the specific neuronal markers NF-L and NSE. The resultsshowed that GFAP, one of the gila markers, was remained no more than4% even after two weeks. The evidence from western blotting analysiswas similar. These data indicated that the most of cultured MSCs weredifferentiated to neuronal cells, whereas not glial cells. The results offlow cytometry showed that undifferentiated MSCs expressed HLA classⅠmolecules in the moderate level, but no HLA classⅡandco-stimulatory molecules were found. With differentiation along neuroallineage, the expressing of HLA classⅠ, classⅡand CD80 significantlyincreased compared with undifferentiated MSCs(P＜0.05). However,even after differentiation for two weeks in vitro, the expression of CD40and CD 86 were no difference, as shown by flow cytometry and westernblot analysis. Afterγ-IFN pre-treatment, the expression of HLA classⅠand classⅡon cells was significantly higher than that on cells of IFN-γuntreated groups. Nevertheless, the results of flow cytometry assay andWestern blot analysis indicated that the expression of CD40, CD80, andCD86 were unaffected by IFN-γ. After 6 days co-culture of MSCs in thecourse of neuronal differentiation and allogenic T cells, the proliferationof T cells was significantly decreased. With differentiation along neuroallineage, the inhibitory action of cells were significantly enhancedConclusion: MSCs in the course of neuronal differentiationincreasely expressed HLA classⅠ, classⅡand CD80, but did not expressCD40 and CD86. Furthermore, these cells suppressed T cell proliferation.Athough immunogenicity of human bone marrow MSCs in the course ofneuronal differentiation remained in a low level, it showed an increasingtendency.PartⅡ: Mesenchymal stem cells in the course of neuroal differentiation suppresse allogenimic T lymphocytes proliferationObjective: To study whether neurocytes induced from bone marrowMSCs could suppresses allogenic T lymphocytes responsiveness, andexplore its possible mechanisms.Methods: Human MSCs isolated and expanded from bone marrowwere cultured in a neuroal medium from one week to two weeks todifferentiat along neuroal cells. The subgroups of cells refered to PartⅠ.Allogenic T lymphocytes were isolated and stimulated byphytohemagglutinin (PHA) in vitro. Cells of each group were added inthe culture system individually. In some experiments, IFN-γ100 U/mlwas added to the culture medium prior to 2 days. To determine whethersuppression of neuronal differentiated MSCs to the T cells proliferation isdependent on cell-cell contact, transwell plates were used. T cells werecultured in the upper chamber of transwell plates. Neuroal differentiatedMSCs were cultured in the lower chamber. T lymphocytes proliferationwas assessed by [~3H]-thymidine incorporation using a liquid scintillationcounter.Results: Untreated MSCs and neuroal differentiated MSCs inhibitedthe proliferation of T cells in response to PHA treatment. Theproliferation of T cells were suppressed: from 67564±1321 cpm for theproliferation of T cells in response to PHA, to 29547±1435～56473±798 cpm for proliferation of T cells in response to PHA in the presence ofundifferentiated MSCs or neuronal differentiated MSCs (P＜0.05). Thesuppression effect of MSCs cultured in the neuronal medium wasincreased. An increase was evident inγ-IFN pre-treatment groupscompared withγ-IFN untreated groups for the proliferation of T cells.(P＜0.05). Compared with cell-cell contact culture between neuroaldifferentiated MSCs and T cells, the suppression effect of transwellculture was not significantly changed (P＞0.05). Conclusion: MSCs in the course of neuroal differentiation inhibitedthe proliferation of T cells in response to PHA treatment. This resultindicated that there was immunomodulatory function of MSCs in thecourse of neuroal differentiation in vitro. The effect of soluble factorswere possible mechanisms.PartⅢ: Immunological rejection induced by the transplantation ofbone marrow MSCs in the course of neuronal differentiation into thebrain of HIBD modelObjective: To study reject reaction induced by the transplantation ofneuronal differentiated bone marrow MSCs into the brain of HIBDmodel.Methods: 7-day-old neonatal Kunming(KM) mice were randomlydivided into five groups: (1) normal control group (Con group, n=12); (2)HIBD model group (HIBD group, n=12); (3) transplantation ofundifferentiated MSCs group (MSCs group, n=12); (4) transplantation ofcells differentiated for one week group (Neu1 group, n=12); (5)transplantation of cells differentiated for two weeks group (Neu1 group,n=12). Hypoxia-ischemia neonatal mouse model were replicatedaccording to Ditelberg(1996). Mice in 10-day-old (the third day afterHIBD) were transplanted with 1×10~5 cells into the brain. Effect oftransplantation was evaluated with a battery of behavioral tests andpathologic analysis. The expression of HLA classⅡ, CD11C and Mac-1were detected with immunofluorescence.Results: HIBD mice which performed the T maze, the radial armmaze, and foot-faults tests were significantly worse than normal mice.While the mice transplanted with MSCs in the course of neuroaldifferentiation performed much better than HIBD mice. There was nosignificantly difference among three groups. Four weeks after HIBD, the mice were put to death, and their brains were obtained. Leftencephalatrophy was found in the brains of HIBD group, MSCs group,Neu1 group and Neu2 group. Nevertheless, obvious abnormality was notfound in the brain of Con group. The mice brains were sliced and stainedwith hematoxylin and eosin. The brain tissue sections were observed bylight microscope. In Con group, cortical neurons were distributedregularly and cells in the hippocamp CA1 district lined up in order. Whilesome of cortical neurons were lost and cells in the hippocamp CA1district lined up chaoticly in HIBD group. The neuron damage in MSCsgroup, Neu1 group and Neu2 group was significantly improved, ascompared with HIBD group. HLA classⅡwas expressed on the surfaceof MSCs in the course neural differentiation after the cells weretransplated into the brains of HIBD mice model. The percentage ofHLA classⅡpositive cells was low in the first week post transplantation,but significantly increased after two weeks and four weeks. Thepercentage of HLA classⅡpositive cells in Neu2 group was higher thanthat in MSCs group. CD11C and Mac-1 was not expressed in Con group.The expression of CD11C and Mac-1 was in a low level in the HIBDgroup. The expression of CD11C and Mac-1 in MSCs group, Neu1 groupand Neu2 group was higher than that in HIBD group. The expression inNeu2 group was the highest.Conclusion: MSCs in the course of neural differentiation cerebraltransplantation can reduce neurological function defects. There was nosignificant difference in the improvement of neurological function defectsbetween MSCs and cells differentiated along neural lineage. Cerebraltransplantation in neonatal mice after HIBD might improve theimmunogenicity of transplanted cells and cause aggregation of dendriticcell and macrophage in brain. Immunological rejection might be inducedin vivo. The main contents and conclusion of this paper were summarized asfollows:1. Athough immunogenicity of human bone marrow MSCs in the courseof neuronal differentiation remained in a low level, it showed atendency of increasing.2. There was immunomodulatory function of MSCs in the course ofneuroal differentiation in vitro.3. The effect of soluble factors were possible mechanisms of T cellinhibitory by neuroal cells differentiated from MSCs.4. MSCs in the course of neuroal differentiation cerebral transplantationcan reduce neurological function defects. There was no significantdifference in the improvement of neurological function defectsbetween transplantation of MSCs and cells differentiated alongneural lineage.5. MSCs in the course of neuroal differentiation cerebral transplantationin neonatal mice after HIBD might improve the immunogenicity oftransplanted cells and cause aggregation of dendritic cell andmacrophage in brain.6. Immunological rejection might be induced in vivo.