Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies

Abstract

Background: Mesenchymal stem cells (MSCs) can be induced to differentiate into neuronal cells under appropriate cellular conditions and transplanted in brain injury and neurodegenerative diseases animal models for neuroregeneration studies. In contrast to the embryonic stem cells (ESCs), MSCs are easily subject to aging and senescence because of their finite ability of self-renewal. MSCs senescence seriously affected theirs application prospects as a promising tool for cell-based regenerative medicine and tissue engineering. In the present study, we established a reversible immortalized mesenchymal stem cells (IMSCs) line by using SSR#69 retrovirus expressing simian virus 40 large T (SV40T) antigen as an alternative to primary MSCs.

Methods: The retroviral vector SSR#69 expressing simian virus 40 large T (SV40T) antigen was used to construct IMSCs. IMSCs were identified by flow cytometry to detect cell surface makers. To investigate proliferation and differentiation potential of IMSCs, cell growth curve determination and mesodermal trilineage differentiation tests were performed. Neuronal differentiation characteristics of IMSCs were detected in vitro. Before IMSCs transplantation, we excluded its tumorigenicity in nude mice firstly. The Morris water maze tests and shuttle box tests were performed five weeks after HIBD models received cells transplantation therapy.

Results: In this study, reversible IMSCs were constructed successfully and had the similar morphology and cell surface makers as primary MSCs. IMSCs possessed better ability of proliferation and anti-senescence compared with primary MSCs, while maintained multilineage differentiation capacity. Neural-like cells derived from IMSCs had similar expressions of neural-specific genes, protein expression patterns and resting membrane potential (RMP) compared with their counterparts derived from primary MSCs. There was no bump formation in nude mice subcutaneously injected with IMSCs. IMSCs played same role as primary MSCs to improve learning ability and spatial memory of HIBD rats.

Conclusions: IMSCs not only retain their features of primary MSCs but also possess the ability of high proliferation and anti-senescence. IMSCs can definitely be induced to differentiate into neuronal cells in vitro and take the place of primary MSCs for cell transplantation therapy without tumorigenesis in vivo. The stable cell line is particularly useful and valuable as an alternative to MSCs in neuronal differentiation and neuroregeneration associated studies.

Figure 1

Morphology and identification of rat primary mesenchymal stem cells (MSCs). A: Morphology of rat primary MSCs at passage 3 (Scale Bar = 100 μm). B: Cultured primary MSCs were stained with haematoxylin-eosin (HE) (Scale Bar = 100 μm). C-H: Primary MSCs (passage five) were analysed by FACS to detect the expression of mesenchymal surface markers using monoclonal antibodies. (C) CD34-fluorescein isothiocyanate (FITC) labeled; (D) CD45-Phycoerythrin (PE) labeled; (E) CD29-FITC labeled; (F) CD44-FITC labeled; (G) CD90-PE labeled; (H) CD106-PE labeled.

Figure 2

Construction, morphology and characteristics of rat immortalized mesenchymal stem cells (IMSCs). A: Schematic drawings of the integrating component of retroviral vector SSR#69 for reversible immortalization. SSR#69 contains the hygromycin B resistance gene (Hyg R) as a positive selectable marker and the herpes simplex virus thymidine kinase gene (HSV-TK) as a negative selectable marker. The SV40T, Hyg R and HSV-TK genes are flanked by loxP sites. B: Morphology of rat IMSCs at 40 passage (Scale Bar = 100 μm). C: Cultured IMSCs were stained with haematoxylin-eosin (HE) (Scale Bar = 100 μm). D: Western bloting was performed to determine the protein level of SV40T antigen in three different kinds of MSCs. SV40T antigen expressed highly in IMSCs, however, it almost could not be detected in IMSCs after Cre-recombination. MSCs did not express SV40T antigen.

Figure 3

Identification of rat immortalized mesenchymal stem cells (IMSCs) by FACS. IMSCs were analysed by FACS to detect the expression of mesenchymal surface markers using monoclonal antibodies. (A) CD34-fluorescein isothiocyanate (FITC) labeled; (B) CD45-Phycoerythrin (PE) labeled; (C) CD29-FITC labeled; (D) CD44-FITC labeled; (E) CD90-PE labeled; (F) CD106-PE labeled.

Figure 4

Proliferation capacity of three different kinds of MSCs and trilineage potential of IMSCs. A: Growth curve of three different kinds of MSCs (primary MSCs, IMSCs, and IMSCs Cre T antigen). The number of viable cells gradually increased from D0 and attained peak at D3 in all three kinds of MSCs. And then, viable cells number decreased sharply from D3 to D5. The viable cells number of IMSCs was significantly more than another two kinds of MSCs from D2 to D5 (*, p < 0.01 by one-way ANOVA). The tendence of cell growth of IMSCs was reversible with cre recombination. B: Osteocytes, chondrocytes and adipocytes were induced from primary MSCs and IMSCs. IMSCs displayed the same mesodermal trilineage differentiation potential as MSCs (Scale Bar = 20 μm).

Figure 5

IMSCs exhibit the same neuronal differentiation capacity as primary MSCs in vitro. A-D: Transcriptional expression of Nestin, NSE, MAP-2, GDNF during neuronal differentiation of MSCs and IMSCs. The transcriptional expression of genes was determined by measuring their mRNA levels with real-time PCR. Treatment of primary MSCs and IMSCs with MNM resulted in a dramatic increase in the mRNA levels (*, P < 0.001 by Student's t test). Real-time PCR results were confirmed in at least three batches of independent experiments with β-actin normalization. E: Fold increase of Nestin, NSE, MAP-2, and GDNF mRNA expression levels after induced by MNM. The mRNA expression levels of neural markers in both MSCs and IMSCs increased about 8, 10, 5 and 3 fold increases after treated with MNM, respectively (P > 0.05 by Student's t test). F: Immunofluorescence staining of Nestin, NSE, MAP-2, and GDNF during neuronal differentiation of MSCs and IMSCs (Scale bar = 20 μm). G: Expression of NSE during a 24 hr span including 0, 6, 12, 18, and 24 hr time-point of MNM induction in both primary MSCs and IMSCs were analyzed by western blotting. Equal loading of the samples was confirmed by comparable levels of β-actin detected in each lane. H: Resting membrane potential was measured by using whole cell patch clamp. The values represented Means ± S.E.M. (*, p < 0.001, MSCs vs. MSCs + MNM; ^&, p < 0.001, IMSCs vs. IMSCs + MNM; p > 0.05, MSCs + MNM vs. IMSCs + MNM, by Student's t test).

Figure 6

Tumorigenicity detection of IMSCs. A: Inoculated rat yolk sac tumor cell lines (L2RYC) performed peanut-sized bumps in injection sites (the left front and rear notum of nude mice), but no bump generated in IMSCs injection sites (the right front and rear notum of nude mice). B: No bump generated in both primary MSCs injection sites (the left front and rear notum of nude mice) and IMSCs injection sites (the right front and rear notum of nude mice). C: Haematoxylin-eosin (HE) staining for tumor nodules derived from L2RYC injection sites (Scale bar = 100 μm).

Figure 7

Therapeutic effects of primary MSCs and IMSCs in rat model of neonatal HIBD by cell transplantation. A: In hidden platform tests (from D2 to D5), the escape latency of PBS group (*, p < 0.01, PBS vs. control) became shorter after primary MSCs or IMSCs therapy (^&, p < 0.05, MSCs/IMSCs vs. PBS). There were no statistical difference between primary MSCs group and IMSCs group (p > 0.05). B: In hidden platform tests (from D2 to D5), the path lengths of PBS group (*, p < 0.01, PBS vs. control) became shorter after primary MSCs or IMSCs therapy (^&, p < 0.05, MSCs/IMSCs vs. PBS). There were no statistical difference between primary MSCs group and IMSCs group (p > 0.05). C: In the probe trial on the sixth day, the passing times of PBS group (*, P < 0.01, PBS vs. control) significantly increased after primary MSCs or IMSCs therapy (^&, P < 0.05, MSCs/IMSCs vs. PBS). And the MSCs group and IMSCs group had similar passing times (P > 0.05). D: The active avoidance response rate (AARR) of the MSCs group and IMSCs group were significantly higher than that of PBS group on the 3rd, 4th and 5th test day (^&, P < 0.05, MSCs/IMSCs vs. PBS). E: The no avoidance response rate (NARR) of MSCs group and IMSCs group were significantly lower than that of PBS group on the 2^nd and 3^rd test day (^&, P < 0.05, MSCs/IMSCs vs. PBS).