Effect Of ILK Expression On Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Into Neural Cells

Objective: Human umbilical cord mesenchymal stem cells (hUCMSCs) are isolated from Wharton's jelly of the umbilical cord which can be obtained easily. UCMSCs have capability of expansion, self-renewal and differentiation into multiple cell lineages including neural cell lineages. These suggest that hUCMSCs have the potential for treatment of neurodegenerative disorders and injurys of nervous system as a novel cell source for cell replacement. However, the mechamisms of the differentiation from MSCs to neural cell are known unclearly.Recently, there are two tendencys on the studys of the differentiation from MSCs to neural cell. One is the directional induction from MSCs to special neural cell such as dopaminergic neuron, the other is the induction from MSCs to neural stem cell-like cells. Protocols for high-yield generation of undifferentiated neural stem cell (NSC)-like cells from bone marrow stromal cell (BMSCs) of primate and human origin were reported. Undifferentiated NSCs are commonly used and are more suitable for neurotransplantation compared with fully differentiated neural cells, as differentiated neural cells are well known to poorly survive detachment and subsequent transplantation procedures. MSC-derived NSC-like cells (MSC-NSCs) provide a powerful tool for investigating the molecular mechanisms of neural differentiation, and might serve as an autologous cell source to treat acute and chronic neurodegenerative diseases.MSC-NSCs grow in suspended neurosphere-like structures and express high levels of early neural stem cell markers such as CD133 and nestin. Therefore, the differentiation procedure from MSCs to neural cells can be classified two stages. The first stage is from MSCs to NSC-like cells. The second stage is from NSC-like cells to neural cells. This complex process may be simplified as a sequential process of MSCs detachment from the extracellular matrix (ECM), reattachment onto new ECM, and final differentiated into new cell lineages. The dynamic interactions between cell-ECM and cell-cell may play critical roles during differentiations. Cell adhesion to the ECM results in differentiation, proliferation, and suppression of apoptosis. Integrin-linked kinase (ILK) is a novel, ankyrin repeat-containing serine threonine kinase that exhibits cell type-dependent activation and inhibition, the activity of which is rapidly and transiently stimulated by cell-ECM interaction. It is an important regulator of cell-ECM inter-action that leads to cell growth adhesion, migration, proliferation, differentiation, spread, survival and death. So we suppose ILK may play an important role during the differentiation from MSCs to neural cells.At present, there are a few studys on the differentiations from hUCMSCs to dopaminergic neurons, no reports on the differentiations from hUCMSCs to NSC-like cells, and no reports about effects of ILK on the differentiations from hUCMSCs to neural cells. Our experiment is to induce hUCMSCs into hucNSCs (neural stem cell-like cells derived from human umbilical cord), and then to induce hucNSCs into neural cells especially dopaminergic neurons in vitro. Moreover, our study is to investigate ILK expression during conversion of hUCMSCs into hucNSCs and neural cells and to explore the effects on the differentiations inhibiting ILK expression by transfectiong of siRNA-ILK.Methods:1 Cell culture and phenotype analyseThe full-term umbilical cords from healthy children were collected and washed in Hanks'balanced salt solution. The mesenchymal tissue (in Wharton's jelly) was then diced into cubes of approximately 0.5 cm3. Mesenchymal tissue was treated with collagenase II at 37°C for 18 hour. The dissociated mesenchymal cells were further dispersed by treatment with 20% FBS-DMEM. Treated with 0.25% trypsin, the 3 passage cells at a concentration of 3.0×106/ml were analyzed by FACS calibur flowcytometry with CD44-PE,CD29-APC,CD105-FITC. The hUCMSC phenotype was confirmed by FACS analysis.2 Induction of hUCMSCs to hucNSCsThe passage 3 hUCMSCs were harvested and transferred to 24-well plate at a density of 1×105/mL. Differentiations were performed using three groups.1 bFGF+EGF+2%B27; 2 bFGF+EGF; 3 bFGF+EGF+B27+1%FBS. EGF and bFGF were supplemented with concentration 20ng/ml. After sphere formation could be observed. These neurosphere-like structures were expanded for an additional 2 weeks, FACS analysis were started to detect CD44,CD29 and CD105 phenotype. Immunocytochemistry was carried out using standard protocols with antibodiesnestin, CD133, NSE, GFAP and TH.3 Conversion of hucNSCs to neural cells and dopaminergic neuron Induction of terminal neural differentiation was initiated by plating the cells on poly-L-lysine coated glass cover slips. The protocols were classified 4 groups by different inducton medium supplement. 1 10%FBS, 2 ATRA, 3 GDNF+IL-1β, 4 ATRA+GDNF+IL-1β. Concentration was follow : GDNF10ng/ml,IL-1β10ng/ml,ATRA1.0μmol/l. Phase-contrast microscope was used to record morphology changes, immuno-fluorescence was applied to view phenotype changes and RT-PCR was used to reflect the levels of mRNA expression of neural markers such as NSE, GFAP and TH.4 Cystoskeleton changesThe hUCMSCs, hucNSCs and NCs (neural cells obtained by induction) on poly-L-lysine coated glass cover slips were altogether prepared. Cystoskeleton changes were detected by AF488 immunostaining.5 ILK express in hUCMSCs, hucNSCs and NCsImmuno-fluorescence, RT-PCR analyses and Real-Time PCR were carried out to display changes of ILK expression in hUCMSC, hucNSC and neural cells.6 ILK siRNA inhibited ILK express in hucNSCsFour specific ILK siRNA duplex was synthesized by JIMAN Inc. of ShangHai based on the sequence of the human ILK gene (GenBank? Accession Number gi3150001). The hucNSC spheres cultured for 2 weeks were treated with 0.01% trypsinization and machinery blow to form sigle cell suspension, then transfected siRNA according to the manufacturer's protocol. Real time PCR was performed to select which sequence of the siRNA-ILK was the best one to interfere ILK express in hucNSCs.7 Proliferation, apoptosis and differentiation of hucNSCs transfected siRNA ILKMTT was used to detect proliferation change of hucNSCs after transfected siRNA ILK for 72 h. After treated with apoptosis kit, FACS displayed cell apoptosis of hucNSCs transfected siRNA ILK. The hucNSCs transfected siRNA ILK were indued by the same protocol (ATRA, GDNF and IL-1β). Immunofluorescence staining was performed to neural markers such as NSE, GFAP and TH express in differenciated cells. The results were campared with differenciated cells of untransfeccted siRNA.Results:1 Cell culture of hUCMSCs and phenotype analyseTwo days after primary culture, adherent cells with a homogenous fibroblastic morphology were observed. Two weeks after plating, when the cells reached 80%～90% confluence, they were detached with 0.25% trypsin solution and were passaged at a ratio of 1: 2 to 1: 3. Several hours after plating, cells became adherent. And 5 days later, cells formed a compact, whirling-like appearance in low power field. After 10 passages, thses cells maintained their homogenous fibroblastic appearance and growth characteristics. FACS revealed that UC-MSCs expressed putative markers of MSCs, which included CD29, CD44 and CD105.2 Induction of hUCMSCs to hucNSCsThe hUCMSCs concentrated to form clump after supplemented with bFGF, EGFs and 2%B27 for 24h, then cell bodys converged in the second day forming a ball with short process. At last, suspension neurosphere-like structures emerged in 72h. Sphere formation could not observed when hUCMSCs were platd in the medium supplemented FBS or in medium without B27. FACS showed hucNSC lose characteristics of MSCs, CD29, CD44,CD105 were negative. However, iimmunofluorescence staining displayed that these cells spheres expressed early neuroectodermal proneural markers such as nestin and CD133 positively.3 hucNSCs differentiated into neural cells.The hucNSCs decompounded gradully when added in 10%FBS medium and grown out cells radiatively. But cell shapes were unlike UCMSC as well as neural cells. Supplemented with induction medium contained IL-1β,GDNF and RA 2-3d, hucNSCs sphere grown out scattering cells with Fusiform shape, someone had long processes. After induced 10 days, hucNSC decompounded completely leaving behind grana-like shadow. The cell bodys shrinked roundly and someone was irregularly with strong refaction. Terminally about 17 days, periphery of cell bodys stretched out many processes connecting each other like net forming neural cell-like structure. Immunofluorescence revealed that hucNSCs can be differentiated into the neural phenotypes that induced by IL-1β, GDNF and RA: GFAP(21.1±3.5%), NSE(57.4±4.3%), TH(9.2%±3.1%), which represent astroglia, neurons and dopaminergic neurons respectively. Compared with other experimental groups, the percent of neural phenotypes were highest. (p<0.05). Cultured in 10%FBS/DMEM medium, only a few differentiated cells expressed GFAP positively. RT-PCR confirmed the neural-like cells derived from hucNSCs expressed GFAP, NSE and TH on mRNA level.4 Cystoskeleton changesCystoskeleton patterns varied from hUCMSCs to those of hucNSCs and neural cells. UCMSCs revealed a silkiness pattern, hucNSCs displayed a nest shape, while neural cells appeared a branching pattern.5 ILK express in hUCMSCs, hucNSCs and NCsDetected by immunofluorescence, ILK expressed negatively in hUCMSCs, hucNSCs expressed a high level of ILK, double staining demonstrated hucNSCs expressed ILK and nestin at same time. RT-PCR shows ILK expression was poor in hUCMSCs but strong in hucNSCs and NCs. ILK mRNA level in hucNSCs was 12 times than that in hUCMSCs, while ILK mRNA in neural cells was 8.6 times than that in hUCMSCs as analyzed by Real-Time PCR(p<0.05).6 Proliferation, apoptosis and differentiation of hucNSCs transfected siRNA ILKThe best sequence of siRNA was selected. Sence: 5'-GAA UCU CAA CCG UAU UCC ATT-3', antisence: 5'-UGG AAU ACG GU U GAG AUU CTG-3'. ILK could be knock down more than 75%.Proliferation of hucNSCs was inhibited by transfecting with siRNA-ILK. (P<0.01). Apoptosis assay by FACS displayed the early phase apoptotic cells were 11.2%, the later phase apoptotic cells were 9.1%. The positive cells of neural markers NSE, GFAP and TH in differentiated cells were obviously decresed when hucNSCs's ILK gene was inhibited by siRNA transfecting. Contrast with the control group, (P<0.01).Conclusions: MSCs derived from human umbilical cord could be easily isolated and cultured in vitro utilizing collagenase digestion method. UCMSCs characterized with fibroblastic morphology, capability of passage, freezing conservation and resuscitation, expressing high level phenotype CD29, CD44 and CD105. UCMSCs could be converted into neural stem cell-like cells induced with serumfree medium supplemented EGF, bFGF and B27. These hucNSC cells grow in neurosphere-like structures, express high levels of early neuroectodermal proneural markers, such as CD133 and nestin, but lose the characteristics of hUCMSC. The hucNSCs also had capabilities of proliferation and passaging. Withdrawing EGF, bFGF and B27 and adding serum medium, The hucNSCs only could be differentiated into a few cells expressing GFAP positively. While induced by IL-1β, GDNF and RA, hucNSCs can be differentiated into the neural phenotypes: astroglia, neurons and dopaminergic neurons. Dopaminergic neurons obtained terminally were average 10%. Cystoskeleton was rearranged and ILK expression was upregulated during the conversion of MSCs to neural cells, siRNA ILK inhibited proliferation of hucNSCs, arised apoptosis and decreased differenciation to neural cells. Therefore,ILK may be involved in promoting formation of hucNSCs and differenciation of hUCMSCs to neural cells.