The Isolation, Analysis And Directed Differentiation Into Dopaminergic Neurons Of Clonal Older Multipotent Adult Progeniter Cells

Stem cells, with their extensive regeneration potential and functional multilineagedifferentiation capacity, are highly attractive for many applications such as tissue engineering,cellular therapies. The most classification of stem cells is including two types: embryonic stemcells and adult stem cells. ESC is derived from the inner cell mass of the blastocyst. ASC arederived from different adult tissues. Stem cells persist throughout life in numerous mammaliantissues, replacing cells lost to homeostatic turnover, injury, and disease. However, stem cellfunction declines with age in a number of tissues, including the blood, forebrain, skeletal muscle,and skin. These declines in stem cell function may contribute to degeneration and dysfunction inaging regenerative tissues. Thus, age-related changes in the function of stem cells and otherprogenitors may contribute to some diseases of aging, particularly in regenerative tissues, evenwhile other diseases of aging may not be influenced by stem cell aging at all.Parkinson’s disease (PD) is a progressive neurodegenerative disease that affects at least1%of the population over55years old. PD is characterized by the degeneration of dopamine neuronsin the substantia nigra, often coupled with the presence of Lewy bodies. The typical symptom ofPD is loss of motor function, which is due to dopamine (DA) neuron dysfunction. The currenttreatment is primarily based on a dopamine replacement strategy using the dopamine pro-druglevodopa, which is complicated by its half-life and dyskinesia. Because there is only one cell typeaffected in a distinct area of the brain in PD, a feasible therapeutic approach is to expand stem cells,induce them to differentiate into new DA neurons, and transplant the mature cells into the patient’sbrain.The limitation of stem cell therapy is that cells for transplantation must be safe and effective.It has been demonstrated that embryonic stem cells (ESCs) can be induced into DA neurons in vitro;however, ESCs can form teratomas in vivo and elicit immune rejection. Neural stem cells (NSCs)could be another treatment option for PD, but these cells can have low differentiation efficiency;additionally, there is a shortage of sources. Recent reports have shown that induced pluripotentstem (iPS) cells from patients themselves have almost the same pluripotency as ESCs and avoidimmune rejection, and iPS cells can be induced into DA neurons in vitro and improve symptoms ofanimals in vivo. However, iPS cells can also form teratomas, and the effects of the ectopicexpression of genes used to derive iPS cells are still unknown.To avoid problems after transplantation, such as immune rejection and malignanttransformation, autologous stem cells may be ideal candidates for cell therapy. Autologous stemcells from bone marrow have been reported in many papers, including mesenchymal stem cells (MSCs), MAPCs, hBMSCs from young donors, MIAMI cells, and others. These autologous stemcells are multipotent because they can differentiate into three germ layer-derived cells, includingcardiomyocytes, DA neurons, and hepatocytes. MSCs have even been reported to haveimmunoregulatory properties. However, little is known about the culture conditions anddifferentiation potential of autologous stem cells from older humans, which is relevant becausePD and Alzheimer’s disease patients are typically older than55.In this report, we compared different culture conditions for stem cells from older humanbone marrow and identified coMAPC that could proliferate in vitro for at least60PDs andexpress pluripotent genes, including Oct4, Sox2, and Nanog. In vitro studies also proved thatcoMAPCs differentiated into three germ layer-derived cell types. Genome CHIP resultsdemonstrated that coMAPC expressed lower levels of aging-related markers than unclonedMAPC, including p16, p19, p21, p53et al. After induction for more than20days in vitro withretinoic acid (RA), basic fibroblast growth factor (bFGF) and sonic hedgehog (SHH) using atwo-step method and withdrawal of serum, we collected a high proportion of DA neurons (up to70%) from induced coMAPCs, and these induced cells secreted DA upon depolarization. Ourresults suggest that coMAPCs may be an available cell source for cell transplantation therapy inneurodegenerative disorders such as PD and demonstrate an efficient means for DA neurondifferentiation.PART IIsolation and culture of clonal aged multipotent adult progenitor cells fromhuman bone marrowMethods: Human bone marrow cells were donated by patients undergoing hip joint replacementoperations (between50-70years old, no gender classification). The BMMNCs (bone marrowmononuclear cells) were harvested after centrifugation and then plated in hMAPC expansion mediumon fibronectin (FN). Expansion medium consisted of60%low-glucose DMEM (Gibco BRL),40%MCDB-201(Sigma),2%FCS (Gibco BRL),1х insulin transferrin selenium (Sigma),1х linoleicacid,1х human serum albumin (HSA),10-9M dexamethasone (Sigma),10-4M ascorbic acid2-phosphate (Sigma),100U penicillin, and1000U streptomycin (Gibco) supplemented with5ng/mlbFGF (R&D),10ng/mL EGF (Gibco BRL) and10ng/mL PDGF-BB (R&D). Clonal culturation: Afterlimiting dilution (1-5cells per well in a96-well plate), wells containing a single cell were selected toisolate purified stem cells. When these cells were grown to40–50%confluence, the cells from each well(representing the progeny of one clone) were passaged in1well of a24-well plate and then seriallyreseeded in6-well plates and petri dishes at a density of4×103to8×103cells/cm2. Osteogenic differentiation:1.5×10-4sodium β-glycerophosphate (Sigma) and1.0×10-9M dexamethasone wereadded to the medium, and cells were cultured for two weeks.. Chondrogenic differentiation A total of1.5×10-4mg/ml LAA and1ng/ml TGF-β were added to the medium, and cells were cultured for twoweeks. Collagen II expression was evaluated using IHC. Adipogenic differentiation A total of1×10-7dexamethasone and1.0×10-10bovine insulin were added to the medium, and cells were cultured for twoweeks. Cells were stained using oil red O, and PPAR-γ expression was determined using IHC.Endodermal differentiation A combination of20ng/ml HGF and10ng/ml FGF4was added to themedium.Results: We used two different isolation methods to expand multipotent stem cells from bonemarrow. We chose Lymphorep as the best method for isolation stem cells derived from aged bonemarrow. When these cells were grown to40–50%confluence, the cells from each well (representing theprogeny of one clone) were passaged. Some proliferating cells showed apoptosis and stopped mitosisduring serial reseeding when they reached a high density (data not shown). coMAPCs were spindle-likemorphologically and exhibited a high nucleus-to-cytoplasm ratio. Clonal coMAPC lines proliferatedstably in vitro and reached more than80population doublings (PDs). PD time was maintained at32hours for at least2months, which was calculated by counting the cell number daily. FACS analysisdemonstrated that coMAPCs expressed high levels of CD29, CD44, and CD90and low levels of CD106.In addition, hMAPCs were negative for CD34and CD45and showed low immunogenicity for HLA-1.coMAPCs cultured for P4and P22from three different clones demonstrated no evidence of increasedploidy, suggesting that euploidy is maintained during culture expansion. Immunofluorescence showedthat coMAPCs were positive for Vimentin and Sma. After induction, cells showed differentiation andexpressed the biomarkers of different lineage including mesodermal, ectodermal and endodermalderivatives.Conclusion: we have identified are a unique population from older bone marrow, which wecelled coMAPC. These mulitopotent adult stem cells, was isolated from total BMMNC culture andcultured with our optimized culture system. coMAPC can proliferate stably without obvious loss ofplasticity. After induction, coMAPC could differentiate into three-germ layer derived cells.PART IIStemness and Aging analysis of single cloned MAPC from senior donators.Methods: coMAPC multipotent makers like Oct4, Oct4A, Oct4B, Sox2and Nanog weredetected by IF and ReverseT-PCR. Total RNA of coMAPC were collected and reverse transcripted intocDNA library. Antisense RNA was transcripted when making cDNA as template. These antisence RNAwere hybrided with Human Genome CHIP U1332.0Plus from Affymetrix. Gene CHIP results were scaned and analysed by Affymetrix Scaner and Affymetrix netservice. Aging cell related genes like p16,p19, p21, p53and hTERT were detected by reverseT-PCR and western blotting.Results: we analyzed the mRNA and protein expression levels of genes known to be associatedwith multipotency. Oct4(including Oct4A and Oct4B), Sox2and Nanog are the classical markers ofpluripotency. Cloned hMAPC D8expressed higher levels of Oct4, Sox2and Nanog. Thus, we chose theclone D8with the highest expression level of multipotent genes for following experiments. Consistentwith the above mRNA level results, the immunofluorescence analyses show that the expression of Oct4,Sox2and Nanog proteins were enriched in the nucleus. Genome CHIP results demonstrated thatcoMAPC D8expressed lower levels of aging-related markers than MSC, PCR and WB test alsotestified coMAPC expressed lower levels of p16, p19, p21, p53, higher level of hTERT.Conclusion: After clonal culturation, hMAPCs expressed genetic markers of ES cells, such asOct4, Sox2, and Nanog, which are believed to be essential for pluripotency. In the same time, clonalhMAPCs showed lower aging markers than unclonally cultured cells. After induction, singlecell-clonedcoMAPCs could differentiate into three derm derived lineage, so clonal coMAPCs got great potentialfor clinical use as seed cells.PART IIIDirected differentiation of coMAPC into dopaminergic neuronsMethods: Dopaminergic neuron Differentiation: The cloned hMAPCs were gradually inducedto dopaminergic neurons in two steps. First, cells in good condition were plated at3000/cm2fordopaminergic neuron differentiation. bFGF (100ng/ml) and RA (10nM) were then added to themedium. Second, after discarding the above medium and washing with PBS, the step two inducersneurobasal (Gibco) and SHH (R&D,250ng/ml) were added to the medium for two weeks. Thedopaminergic neuron markers Nestin, Tuj1, GFAP, and TH were detected using IHC. DopamineEnzyme-Linked Immunosorbent Assay: DA levels were quantitated using an enzyme-linkedimmunosorbent assay (ELISA) kit (Rapidbio, CO) according to the manufacturer’s instructions. Afterdifferentiation, the culture medium was replaced with a balanced saline. The saline solution does notcontain any ingredients that would react with the antibodies. For Ca2+-free SES, Ca2+was substitutedwith4mM magnesium and1mM EGTA. Cells were treated with high-K+solution (56mM KCl) toinduce depolarization. Cells were treated for15minutes at37°C. Samples of100μl were collected andimmediately used for DA quantitation using ELISA.Results: we tried a novel induction method to generate DA neurons from coMAPCs with ahigher efficiency. Using a multistep lineage protocol using bFGF, RA and SHH, we inducedcoMAPCs to DA neurons with an efficiency of almost70%. After the first seven-day step, the combination of bFGF and RA induced coMAPCs to the neuroectodermal lineage, which was shownbecause the multipotent gene marker Oct4decreased and Sox2, which is highly expressed in neuralprogenitor cells, increased. Then, at the second14-day step, SHH, an important gene in DA neurondevelopment induced the neural coMAPCs to differentiate into DA neurons. After the addition of thecytokine SHH seven days later, the induced cells expressed increasing amounts of the DA neuronmarker TH. hMAPCs induced for21days constitutively released DA into the extracellular medium.Interestingly, under depolarizing conditions induced by elevated levels of extracellular K+(56mM),there was no further increase in the constitutive release of DA. This suggests that the constitutiverelease of DA by hMAPCs induced for21days is mediated by neuronal activity. Furthermore, therelease of DA was dependent on extracellular Ca2+because the treatment of cells with Ca2+-freemedium blocked DA release. This result is entirely consistent with the effect of depolarizingconditions on DA release.Conclusion: We showed that DA neurons induced using our two-step method expressedspecific markers and were able to secrete DA upon depolarization. Finally, we demonstrated that DAsecretion of induced DA neurons was consistent with neuronal activity.