Open Access

Induction of mesenchymal stem cell‑like transformation in rat primary glial cells using hypoxia, mild hypothermia and growth factors

  • Authors:
    • Huiping Wei
    • Wenyun Zhou
    • Guozhu Hu
    • Chunhua Shi
  • View Affiliations

  • Published online on: December 7, 2020     https://doi.org/10.3892/mmr.2020.11760
  • Article Number: 121
  • Copyright: © Wei et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

The transformation of rat primary glial cells into mesenchymal stem cells (MSCs) is intriguing as more seed cells can be harvested. The present study aimed to evaluate the effects of growth factors, hypoxia and mild hypothermia on the transformation of primary glial cells into MSCs. Rat primary glial cells were induced to differentiate by treatment with hypoxia, mild hypothermia and basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Immunohistochemistry and western blotting were then used to determine the expression levels of glial fibrillary acidic protein (GFAP), nestin, musashi‑1, neuron specific enolase (NSE) and neuronal nuclei (NeuN), in each treatment group. bFGF and EGF increased the proportion of CD44+ and CD105+ cells, while anaerobic mild hypothermia increased the proportion of CD90+ cells. The combination of bFGF and EGF, and anaerobic mild hypothermia increased the proportion of CD29+ cells and significantly decreased the proportions of GFAP+ cells and NSE+ cells. Treatment of primary glial cells with bFGF and EGF increased the expression levels of nestin, Musashi‑1, NSE and NeuN. Anaerobic mild hypothermia increased the expression levels of Musashi‑1 and decreased the expression levels of NSE and NeuN in glial cells. The results of the present study demonstrated that bFGF, EGF and anaerobic mild hypothermia treatments may promote the transformation of glial cells into MSC‑like cells, and that the combination of these two treatments may have the optimal effect.

Introduction

Cerebral ischemia and the hypoxia that arises from this condition are the direct causes of stroke and other cerebral diseases (1). Over recent years, mild hypothermia therapy and stem cell therapy have been introduced as a treatment option for patients suffering from these conditions (2). For example, randomized, double-blind, Phase III clinical trials have been carried out by two European groups to investigate the potential effects of hypothermia therapy over 6 months, demonstrating that treatment involving mild hypothermia resulted in good neurological recovery (3,4). Additionally, experiments of cerebral infarction in a rat model demonstrated that mild hypothermia treatment can decrease the area of infarction and achieve good levels of recovery in terms of nerve function (5). In addition, there have been several successful cases of stem cell therapy and mild hypothermia combined with stem cell therapy for stroke in infants (68).

A previous study has suggested that basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) can promote the transformation of bone marrow-derived mesenchymal stem cells (MSCs) or adipose MSCs into neural cells (9). Glial cells are primary cells that can be isolated and cultured from the cerebral cortex; these represent a heterogeneous mixture of cell types that have the ability to proliferate and differentiate (10). The development of new induction methods to improve the efficiency of glial cell transformation into neural stem cells, MSCs would allow us to produce greater numbers of specific cells for neural repair treatments.

Previous studies injected the brains of rats with MSCs and achieved good levels of repair in the central nervous system (11,12). In addition, a range of efficient methods have now been described to facilitate the acquisition of mesenchymal stem cells; these cells are important as they have the ability to differentiate into neural cells (13,14). However, a previous study investigated the transformation of rat primary glial cells into MSCs (15). The present study hypothesized that treatment with mild hypothermia or growth factors may promote the differentiation of glial cells into MSCs, thus supplementing nerve cells that died due to hypoxia. The aim of the present study was to use a rat model to investigate the effects of cytokine induction and anaerobic mild hypothermia on the differentiation of primary glial cells.

Materials and methods

Primary isolation of rat glial cells

All animal procedures were approved by the Ethics Committee of Jiangxi Provincial People's Hospital Affiliated to Nanchang University (approval no. 2017BBG70066; Nanchang, China). All experiments involving animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (16). A total of ten specific pathogen-free neonatal Sprague-Dawley rats (1–3 days postnatal; 6–8 g; male:female, 1:1) were provided by the Animal Center of Jiangxi Provincial People's Hospital Affiliated to Nanchang University. The animals were housed in a specific pathogen-free environment at a temperature of 23±2°C, with 45–65% humidity, 12-h light/dark cycles, and free access to food and water. The animals were sacrificed by cervical dislocation and immersed in 75% alcohol for 2–3 min on an ultra-clean table. D-Hank's solution (Gibco; Thermo Fisher Scientific, Inc.), containing a mixture of penicillin (10,000 U/ml) and streptomycin (10 mg/ml), was added to four petri dishes (labeled 1–4). First, the sterilized rats were transferred to dish number 1. Their heads were removed and placed in dish number 2. The skulls were then separated at the brainstem, and the brain was quickly transferred to dish number 3. The cortex was then freed and the meninges on the cortex were fully removed with tweezers and transferred to dish number 4. The cortical tissue was then rinsed carefully in D-Hank's solution and then transferred to a vial containing penicillin (10,000 U/ml) and D-Hank's solution. The tissue suspension was then cut into tissue fragments that were ~1 mm3 in size and then transferred to a centrifuge tube. An equivalent volume of 0.25% trypsin was added to the centrifuge tube, the contents were mixed carefully and then allowed to digest at 37°C for 15 min. The digestion was terminated by adding three equivalent volumes of complete culture medium including DF12 (cat. no. 1859228; Gibco; Thermo Fisher Scientific, Inc.) and 20% fetal bovine serum (FBS; cat. no. 04-007-1A; Biological Industries). The cell suspension was then mixed by gentle blowing with a suction tube and filtered with a 200-mesh, and the isolated cells were centrifuged at 850 × g for 3 min at room temperature. The supernatant was discarded and the cell precipitation was suspended in DF12 medium with 20% FBS. Finally, the cells were cultured with 5% CO2 at 37°C until the first passage prior to subsequent experimentation.

Hypoxia, mild hypothermia and growth factor treatments

Cells at 70% confluency were divided into eight groups for culture in normal culture medium (DF12 with 20% FBS) as follows: Control-normal (astrocytes cultured at 37°C with 5% CO2); control-hypothermia (33°C, 5% CO2, 12 h); control-hypoxia (37°C, 95% N2, 5% CO2, 12 h); control-hypothermia + hypoxia; bFGF + EGF-normal; bFGF + EGF-hypothermia (33°C, 5% CO2, 12 h); bFGF + EGF-hypoxia (37°C, 95% N2, 5% CO2, 12 h); and bFGF + EGF-hypothermia + hypoxia. Cells in the bFGF + EGF groups received 10 ng/ml bFGF (cat. no. P1020-500; Novoprotein) and 20 ng/ml EGF (cat. no. REGFP-05011; Cyagen Biosciences, Inc.) for 7 days. Hypothermia involved a reduction in the culture temperature from 37°C to 33°C for 12 h. To mimic a hypoxic environment, cells were cultured in 95% N2 + 5% CO2 for 12 h. The cells were treated for a total of 7 days and collected for subsequent experiments.

Immunocytochemical staining

Cells were fixed in 4% paraformaldehyde at room temperature for 1 h. Following antigen retrieval by heating in a microwave in Tris/EDTA (pH 9.0), 3% hydrogen peroxide was used to block endogenous peroxidase activity for 15 min at room temperature. After non-specific blocking in 5% BSA (HyClone; GE Healthcare Life Science) at room temperature for 2 h, the cells were incubated overnight at 4°C with primary antibodies against nestin (cat. no. OM264981; OmniAb; 1:250), glial fibrillary acidic protein (GFAP; cat. no. ab33922; Abcam; 1:500), neuronal nuclei (NeuN; cat. no. ab177487; Abcam; 1:500), Musashi-1 (cat. no. bs-20241r; BIOSS; 1:250) and neuron specific enolase (NSE; cat. no. bs-10445r; BIOSS; 1:500). The following morning, the cells were washed with PBS and then incubated with an HRP-conjugated goat anti-rabbit IgG H&L secondary antibody (cat. no. ab6721; Abcam; 1:500) at 37°C for 30 min. Subsequently, the cells were washed with PBS, and stained with 3,3′-diaminobenzidine for 5–10 min at room temperature. Cells were then re-washed in PBS for 1 min and stained with hematoxylin for 3 min at room temperature; this staining was then converted to a blue color using ethanol hydrochloride. Finally, cells were observed by light microscopy. ImagePro Plus (version 6.0; Media Cybernetics, Inc.) was used to measure the mean optical density of positive cells within three randomly selected fields of view at high magnification (×200).

Flow cytometry

Instrument parameters were adjusted by fluorescence homology control. Cells were washed twice with PBS and digested with 0.25% trypsin containing 0.02% EDTA. The cell suspension was then transferred into a 10-ml centrifuge tube. Cells were collected from each treatment group by centrifugation at 850 × g for 3 min at room temperature. Next, 1 ml of PBS solution was added to each tube and centrifuged at 1,700 × g for 1 min at room temperature. The supernatant was discarded, and 5×105 cells were collected in 300 ml PBS. Subsequently, 300 µl 2X binding buffer and 5 µl of the following antibodies were added to each tube: Mouse anti-CD90-FITC (cat. no. 561973; BD Biosciences; 1:100), mouse anti-CD29-PE (cat. no. 102207; BioLegend, Inc.; 1:100), mouse anti-CD44-PE-cy7 (cat. no. ab4679; Abcam; 1:100) and mouse anti-CD105-APC (cat. no. 17-1051-80; eBioscience; Thermo Fisher Scientific, Inc.; 1:100). The tubes were mixed carefully and incubated at room temperature in the dark for 10 min. Multichannel fluorescence counting was then performed on a FACSCalibur flow cytometer (BD Biosciences). The combinations of CD105/CD90 and CD29/CD44 were used in the flow cytometry experiments. Data were analyzed using FlowJo software (version 7.6; FlowJo, LLC).

Western blotting

Cells from each treatment group were mixed with RIPA solution (Beyotime Institute of Biotechnology) and incubated at 4°C for 30 min to create a lysed suspension containing protein extract. The extracts were then centrifuged at 8,500 × g for 10 min at 4°C; the supernatant, containing the total protein extract, was retained for analysis. Next, a bicinchoninic acid kit (Beyotime Institute of Biotechnology) was used to determine the concentration of each protein extract. Proteins (20 µg) were then separated by 10% SDS-PAGE, and then transferred to PVDF membranes. After blocking in 5% skimmed milk at room temperature for 2 h, membranes were then incubated overnight at 4°C with a range of primary antibodies, including: Nestin (cat. no. OM264981; OMNIAB; 1:250), GFAP (cat. no. ab33922; Abcam; 1:500), NeuN (cat. no. ab177487; Abcam; 1:500), Musashi-1 (cat. no. bs-20241r: BIOSS; 1:250), NSE (cat. no. bs-10445r; BIOSS; 1:500) and GAPDH (cat. no. ab8245; Abcam; 1:1,000). The following morning, the membranes were washed with PBS and incubated with an HRP goat anti-rabbit IgG H&L secondary antibody (cat. no. ab6721; Abcam; 1:500) or HRP goat anti-mouse IgG H&L secondary antibody (cat. no. ab6789; Abcam; 1:500) at room temperature for 1–2 h. The membranes were then developed using ECL exposure solution (cat. no. SW2010-1; Beijing Solarbio Science & Technology Co., Ltd.). Quantity one software (version 4.6; Bio-Rad Laboratories, Inc.) was used to analyze the gray value of specific bands of interest.

Statistical analysis

Data are presented as the mean ± SD of six repeats. Comparisons among multiple groups were analyzed using two-way ANOVA followed by a Bonferroni post-hoc test. All data were analyzed using SPSS (version 19.0; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference

Results

Glial cell identification

Data relating to the immunohistochemical detection of GFAP are shown in Fig. 1. Primary cultured glial cells expressed GFAP, indicating that these cells exhibited low levels of differentiation. Some of these cells expressed strong immunofluorescence for GFAP; these cells were polygonal in shape and were bifurcated, thus indicating that they were astrocytes. However, ~40% cells in the staining were GFAP cells, which were small in size. These cells may not be in healthy conditions.

Identification of differentiation clusters by flow cytometry

Flow cytometry was used to detect clusters of differentiated cells (CD44, CD29, CD90 and CD105) associated with MSCs. Under normal conditions, bFGF and EGF treatment increased the proportion of CD105+ cells from <1 to ~30% (P<0.05; Figs. 2 and 3A), and also significantly increased the proportion of CD44+ and CD90+ cells compared with the control group (P<0.05; Figs. 2, 3B and C). However, the proportion of CD29+ cells significantly decreased from ~60 to ~40% following bFGF and EGF treatment compared with the control group under normal conditions (P<0.05; Figs. 2 and 3D). The combined treatment of anaerobic mild hypothermia, bFGF and EGF led to an increase in the proportion of CD29+ cells to ~60% compared with the control group (P<0.05; Figs. 2 and 3D). Additionally, anaerobic mild hypothermia treatment increased the proportion of CD44+, CD90+ and CD105+ cells compared with the normal group (P<0.05; Figs. 2 and 3A-C), although anaerobic mild hypothermia treatment with bFGF and EGF did not show a synergistic effect with regards to increasing the proportion of CD44+ and CD90+ clusters compared with the normal group (P>0.05; Figs. 2 and 3A-C).

Growth factor and anaerobic mild hypothermia treatment decreases the expression levels of NSE, NeuN and GFAP, but promotes the expression levels of nestin and Musashi-1

Cells from the control-normal group and the bFGF + EGF-hypoxia + hypothermia group were tested by immunocytochemistry to determine the expression levels of a range of marker genes that are specific for neural cells, including GFAP, nestin, musashi-1, NSE and NeuN (Fig. 4). Compared with normal cultured glial cells, cells that were treated with anaerobic mild hypothermia, bFGF and EGF demonstrated a decrease in the proportions of GFAP+, NSE+ and NeuN+ cells, but an increase in the proportions of Musashi-1+ and Nestin+ cells; additionally, these cells tended to grow flat on the wall of culture dishes with fewer clusters (Fig. 4).

Western blotting was performed to detect specific cellular markers. Growth factor treatment (bFGF and EGF) led to an increase in the expression levels of nestin and Musashi-1, and a decrease in the expression levels of NSE and NeuN under normal conditions compared with control cells (Figs. 5 and 6). Anaerobic mild hypothermia treatment increased the expression levels of Musashi-1, but decreased the expression levels of NSE and NeuN in glial cells compared with the normal group. Additionally, anaerobic mild hypothermia treatment significantly decreased GFAP expression compared with the control-normal group (P<0.05; Figs. 5 and 6). Additionally, the combination of growth factors and anaerobic mild hypothermia treatments had synergistic effects on the inhibition of GFAP when compared with the bFGF + EGF + normal group (P<0.05; Figs. 5 and 6).

Discussion

According to the classical biological view, cell differentiation is unidirectional; in normal tissues, stem cells can differentiate into a range of different cell types, such as neurons and glial cells (4). Although cells can be induced to differentiate into stem cells by a range of artificial techniques (17), it appears that under normal physiological conditions, differentiated cells cannot revert back to stem cells. However, this view has been challenged. A previous study found that glial cells in dental pulp can undergo a form of ‘reverse differentiation’ and revert back into stem cells (15). This discovery not only challenged the traditional concept, but also created a new option for the treatment of disease. The current study identified differences between growth factor treatment and mild hypothermia treatment with regards to the induction of glial cell differentiation, and that the combination of growth factor treatment and mild hypothermia treatment could improve the proportion of stem cell-like cells derived from a glial cell population.

Neuronal lineage markers and CD molecules were selected as indicators to evaluate the differentiation of glial cells under a range of different treatments involving growth factors, anaerobic conditions and mild hypothermic conditions. Nestin is known to be predominantly expressed in neural stem cells, but not in mature neurons; therefore, nestin is an effective marker for neural stem cells (18). Neuroblasts (neuroprogenitors) are monopotent stem cells with a certain potential for differentiation; these cells differentiate mainly into neurons, astrocytes and oligodendrocytes (19). GFAP, an established marker for neuroblasts, is also expressed in astrocytes (20) and is therefore commonly used to identify neuroblasts containing nestin. Musashi-1, an RNA-binding protein, is mainly expressed in mitotic neural stem cells and is therefore used to identify neural stem cells and neuroblasts (21). NeuN antigen is a nucleoprotein that serves a vital role in neurons (22); the positive expression of NeuN antigen indicates that neurons are no longer undergoing mitotic events (23). NSE is a cytoplasmic protein that is mainly expressed in mature neurons and also represents an effective marker for differentiated neurons (24). The detection of CD molecules is an effective method with which to identify MSCs (25). According to the International Society for Cellular Therapy, the simplest identification criterion for human MSCs is the presence of adherent human cells that exhibit positivity for CD90/Thy1 and CD105/Endoglin, as determined by flow cytometry (26,27). However, this simple criterion cannot be readily applied to the CDs of non-human MSCs. For example, MSCs from mouse adipose tissue can be CD105, and MSCs from rat bone marrow can be CD90 (28). Therefore, the identification of mouse-derived MSCs should also consider positivity for CD29 and CD44 (29,30).

The indicators selected for the present study provide information from two different perspectives. One perspective is to reflect the growth of MSCs in the nervous system by detecting the positivity of MSC transforming clusters on the surface of glial cells in response to different treatments. The other perspective is to measure the expression levels of genetic and protein markers in glial cells in response to different treatment conditions. The central nervous system also contains MSCs; when a large number of neural cells die, MSCs proliferate and then differentiate to help repair the nervous system injury (31). This is the theoretical basis of stem cell therapy, in which MSCs are injected into the brain. Growth factor induction is a common and effective way of promoting this proliferation and to increase the proportion of MSCs produced (29). In order to identify MSCs in an accurate manner, it is important to maintain a high ratio of positivity in specific CD clusters (32). In the present study, CD105+ cells accounted for <0.5% of all glial cells in the normal state; this increased to ~1% after hypoxia and mild hypothermia treatments, thus indicating that mild hypothermia treatments can slightly increase the proportion of MSCs after hypoxia. Additionally, the present study demonstrated that this treatment increased the proportions of cells that were positive for CD105 and CD90 (to 30 and 20%, respectively), although the proportion of cells that were positive for CD44 and CD90 did not change significantly. This indicated that growth factors may induce the proliferation of cells that are positive for both CD105 and CD29. However, these cells may not be MSCs; instead, these cells may be somatic cells exhibiting greater levels of differentiation. The proportion of MSCs that were positive for CD90 was ~15%. This is in accordance with the present immunocytochemistry results, which demonstrated that glial cells in the group receiving growth factors adopted a morphology that was more similar to fibroblasts.

During normal body function, different generations of cells must undergo processes that turn mature cells into stem cells (33). Although research relating to the induction of stem cells by acid has been questioned recently (34), there is no essential difference between the basic concept of this research and stem cell induction. The present study demonstrated that changes in the cellular environment may induce mature cells to become stem cells, although the identity of these specific environmental factors remains unknown. The results of the present study demonstrated that hypoxic mild hypothermia treatment or growth factor treatment upregulated the expression levels of nestin and Musashi-1 in differentiated neurons and downregulated the expression levels of NSE and NeuN in mature neurons. Hypoxia treatment, combined with mild hypothermia and growth factor treatment, further enhanced this effect. Additionally, the results of the present study demonstrated that the relative expression levels of GFAP were inhibited by mild hypothermia or growth factor treatment after hypoxia treatment. It is possible that neural stem cells are positive for GFAP, nestin and Musashi-1, whereas neuroblasts are positive for nestin and Musashi-1 (35). The observed decrease in GFAP expression may indicate that neural stem cells that are derived from MSCs have the ability to rapidly differentiate into nerve cells.

Previous studies have demonstrated that it is difficult to identify MSCs from various rat tissues using surface markers alone (36,37). In the present study, nestin and Musashi-1 were selected as biomarkers for MSCs (38) and the induction of MSC-like transformation in rat primary glial cells by hypoxia, mild hypothermia and growth factors from the morphological aspect was verified. Moreover, CD29 and CD44 positivity supported the MSC-like characteristics, at least to some extent. However, these biomarkers and surface markers are not sufficient to prove the specific features of MSCs. Future work should aim to stimulate transformed cells into different types of neurons (39), which could provide further support for the hypothesis of the current study.

In conclusion, the results of the present study suggested that treatments involving bFGF and EGF, and anaerobic mild hypothermia may promote the transformation of glial cells into MSC-like cells, and that these effects were optimized when the two treatments were combined.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

HW, WZ and GH performed the experiments and analyzed the data. HW and CS designed the study and wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Jiangxi Provincial People's Hospital Affiliated to Nanchang University (Nanchang, China; approval no. 2017BBG70066).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Lee RHC, Lee MHH, Wu CYC, Silva ACE, Possoit HE, Hsieh TH, Minagar A and Lin HW: Cerebral ischemia and neuroregeneration. Neural Regen Res. 13:373–385. 2018. View Article : Google Scholar : PubMed/NCBI

2 

Sun YJ, Zhang ZY, Fan B and Li GY: Neuroprotection by therapeutic hypothermia. Front Neurosci. 13:5862019. View Article : Google Scholar : PubMed/NCBI

3 

Wassink G, Gunn ER, Drury PP, Bennet L and Gunn AJ: The mechanisms and treatment of asphyxial encephalopathy. Front Neurosci. 8:402014. View Article : Google Scholar : PubMed/NCBI

4 

Shankaran S: Outcomes of hypoxic-ischemic encephalopathy in neonates treated with hypothermia. Clin Perinatol. 41:149–159. 2014. View Article : Google Scholar : PubMed/NCBI

5 

Darwazeh R and Yan Y: Mild hypothermia as a treatment for central nervous system injuries: Positive or negative effects. Neural Regen Res. 8:2677–2686. 2013.PubMed/NCBI

6 

Park WS, Sung SI, Ahn SY, Yoo HS, Sung DK, Im GH, Choi SJ and Chang YS: Hypothermia augments neuroprotective activity of mesenchymal stem cells for neonatal hypoxic-ischemic encephalopathy. PLoS One. 10:e01208932015. View Article : Google Scholar : PubMed/NCBI

7 

Savitz SI, Cramer SC, Wechsler L and Consortium S: Stem cells as an emerging paradigm in stroke 3: Enhancing the development of clinical trials. Stroke. 45:634–639. 2014. View Article : Google Scholar : PubMed/NCBI

8 

Cox CS Jr, Hetz RA, Liao GP, Aertker BM, Ewing-Cobbs L, Juranek J, Savitz SI, Jackson ML, Romanowska-Pawliczek AM, Triolo F, et al: Treatment of severe adult traumatic brain injury using bone marrow mononuclear cells. Stem Cells. 35:1065–1079. 2017. View Article : Google Scholar : PubMed/NCBI

9 

Ullah I, Subbarao RB and Rho GJ: Human mesenchymal stem cells-current trends and future prospective. Biosci Rep. 35:e001912015. View Article : Google Scholar : PubMed/NCBI

10 

Kriegstein A and Alvarez-Buylla A: The glial nature of embryonic and adult neural stem cells. Annu Rev Neurosci. 32:149–184. 2009. View Article : Google Scholar : PubMed/NCBI

11 

van Velthoven CT, Kavelaars A, van Bel F and Heijnen CJ: Repeated mesenchymal stem cell treatment after neonatal hypoxia-ischemia has distinct effects on formation and maturation of new neurons and oligodendrocytes leading to restoration of damage, corticospinal motor tract activity, and sensorimotor function. J Neurosci. 30:9603–9611. 2010. View Article : Google Scholar : PubMed/NCBI

12 

Parolini O, Alviano F, Bergwerf I, Boraschi D, De Bari C, De Waele P, Dominici M, Evangelista M, Falk W, Hennerbichler S, et al: Toward cell therapy using placenta-derived cells: Disease mechanisms, cell biology, preclinical studies, and regulatory aspects at the round table. Stem Cells Dev. 19:143–154. 2010. View Article : Google Scholar : PubMed/NCBI

13 

Liu L and Ho C: Mesenchymal stem cell preparation and transfection-free ferumoxytol labeling for MRI cell tracking. Curr Protoc Stem Cell Biol. 43:2B.7.1–2B.7.14. 2017. View Article : Google Scholar

14 

Liu L, Tseng L, Ye Q, Wu YL, Bain DJ and Ho C: A new method for preparing mesenchymal stem cells and labeling with ferumoxytol for cell tracking by MRI. Sci Rep. 6:262712016. View Article : Google Scholar : PubMed/NCBI

15 

Luo L, He Y, Wang X, Key B, Lee BH, Li H and Ye Q: Potential roles of dental pulp stem cells in neural regeneration and repair. Stem Cells Int. 2018:17312892018. View Article : Google Scholar : PubMed/NCBI

16 

National Research Council: Guide for the Care and Use of Laboratory Animals. 8th edition. National Academies Press; Washington, DC: 2011

17 

Donovan PJ and de Miguel MP: Turning germ cells into stem cells. Curr Opin Genet Dev. 13:463–471. 2003. View Article : Google Scholar : PubMed/NCBI

18 

Homem CC, Repic M and Knoblich JA: Proliferation control in neural stem and progenitor cells. Nat Rev Neurosci. 16:647–659. 2015. View Article : Google Scholar : PubMed/NCBI

19 

Jefferis GS and Livet J: Sparse and combinatorial neuron labelling. Curr Opin Neurobiol. 22:101–110. 2012. View Article : Google Scholar : PubMed/NCBI

20 

Song Z, Shen F, Zhang Z, Wu S and Zhu G: Calpain inhibition ameliorates depression-like behaviors by reducing inflammation and promoting synaptic protein expression in the hippocampus. Neuropharmacology. 174:1081752020. View Article : Google Scholar : PubMed/NCBI

21 

Mirsadeghi S, Shahbazi E, Hemmesi K, Nemati S, Baharvand H, Mirnajafi-Zadeh J and Kiani S: Development of membrane ion channels during neural differentiation from human embryonic stem cells. Biochem Biophys Res Commun. 491:166–172. 2017. View Article : Google Scholar : PubMed/NCBI

22 

Zhu G, Wang Y, Li J and Wang J: Chronic treatment with ginsenoside Rg1 promotes memory and hippocampal long-term potentiation in middle-aged mice. Neuroscience. 292:81–89. 2015. View Article : Google Scholar : PubMed/NCBI

23 

Gusel'nikova VV and Korzhevskiy DE: NeuN As a neuronal nuclear antigen and neuron differentiation marker. Acta Naturae. 7:42–47. 2015. View Article : Google Scholar : PubMed/NCBI

24 

Sarnat HB: Clinical neuropathology practice guide 5–2013: Markers of neuronal maturation. Clin Neuropathol. 32:340–369. 2013. View Article : Google Scholar : PubMed/NCBI

25 

Pittenger MF, Discher DE, Peault BM, Phinney DG, Hare JM and Caplan AI: Mesenchymal stem cell perspective: Cell biology to clinical progress. NPJ Regen Med. 4:222019. View Article : Google Scholar : PubMed/NCBI

26 

Lavezzi AM, Corna MF and Matturri L: Neuronal nuclear antigen (NeuN): A useful marker of neuronal immaturity in sudden unexplained perinatal death. J Neurol Sci. 329:45–50. 2013. View Article : Google Scholar : PubMed/NCBI

27 

Galipeau J and Krampera M: The challenge of defining mesenchymal stromal cell potency assays and their potential use as release criteria. Cytotherapy. 17:125–127. 2015. View Article : Google Scholar : PubMed/NCBI

28 

Lv FJ, Tuan RS, Cheung KM and Leung VY: Concise review: The surface markers and identity of human mesenchymal stem cells. Stem Cells. 32:1408–1419. 2014. View Article : Google Scholar : PubMed/NCBI

29 

Zhu H, Guo ZK, Jiang XX, Li H, Wang XY, Yao HY, Zhang Y and Mao N: A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc. 5:550–560. 2010. View Article : Google Scholar : PubMed/NCBI

30 

Sági B, Maraghechi P, Urbán VS, Hegyi B, Szigeti A, Fajka-Boja R, Kudlik G, Német K, Monostori E, Gócza E and Uher F: Positional identity of murine mesenchymal stem cells resident in different organs is determined in the postsegmentation mesoderm. Stem Cells Dev. 21:814–828. 2012. View Article : Google Scholar : PubMed/NCBI

31 

Nandoe Tewarie RS, Hurtado A, Bartels RH, Grotenhuis A and Oudega M: Stem cell-based therapies for spinal cord injury. J Spinal Cord Med. 32:105–114. 2009. View Article : Google Scholar : PubMed/NCBI

32 

Maleki M, Ghanbarvand F, Reza Behvarz M, Ejtemaei M and Ghadirkhomi E: Comparison of mesenchymal stem cell markers in multiple human adult stem cells. Int J Stem Cells. 7:118–126. 2014. View Article : Google Scholar : PubMed/NCBI

33 

Shyh-Chang N and Ng HH: The metabolic programming of stem cells. Genes Dev. 31:336–346. 2017. View Article : Google Scholar : PubMed/NCBI

34 

Kim EM, Manzar G and Zavazava N: Induced pluripotent stem cell-derived gamete-associated proteins incite rejection of induced pluripotent stem cells in syngeneic mice. Immunology. 151:191–197. 2017. View Article : Google Scholar : PubMed/NCBI

35 

Campbell JG, Miller DC, Cundiff DD, Feng Q and Litofsky NS: Neural stem/progenitor cells react to non-glial cns neoplasms. SpringerPlus. 4:532015. View Article : Google Scholar : PubMed/NCBI

36 

Sullivan MO, Gordon-Evans WJ, Fredericks LP, Kiefer K, Conzemius MG and Griffon DJ: Comparison of mesenchymal stem cell surface markers from bone marrow aspirates and adipose stromal vascular fraction sites. Front Vet Sci. 2:822016. View Article : Google Scholar : PubMed/NCBI

37 

Lin CS, Xin ZC, Dai J and Lue TF: Commonly used mesenchymal stem cell markers and tracking labels: Limitations and challenges. Histol Histopathol. 28:1109–1116. 2013.PubMed/NCBI

38 

Xie L, Zeng X, Hu J and Chen Q: Characterization of nestin, a selective marker for bone marrow derived mesenchymal stem cells. Stem Cells Int. 2015:7620982015. View Article : Google Scholar : PubMed/NCBI

39 

Khalil W, Tiraihi T, Soleimani M, Baheiraei N and Zibara K: Conversion of neural stem cells into functional neuron-like cells by MicroRNA-218: Differential expression of functionality genes. Neurotox Res. 38:707–722. 2020. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

February-2021
Volume 23 Issue 2

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Wei H, Zhou W, Hu G and Shi C: Induction of mesenchymal stem cell‑like transformation in rat primary glial cells using hypoxia, mild hypothermia and growth factors. Mol Med Rep 23: 121, 2021.
APA
Wei, H., Zhou, W., Hu, G., & Shi, C. (2021). Induction of mesenchymal stem cell‑like transformation in rat primary glial cells using hypoxia, mild hypothermia and growth factors. Molecular Medicine Reports, 23, 121. https://doi.org/10.3892/mmr.2020.11760
MLA
Wei, H., Zhou, W., Hu, G., Shi, C."Induction of mesenchymal stem cell‑like transformation in rat primary glial cells using hypoxia, mild hypothermia and growth factors". Molecular Medicine Reports 23.2 (2021): 121.
Chicago
Wei, H., Zhou, W., Hu, G., Shi, C."Induction of mesenchymal stem cell‑like transformation in rat primary glial cells using hypoxia, mild hypothermia and growth factors". Molecular Medicine Reports 23, no. 2 (2021): 121. https://doi.org/10.3892/mmr.2020.11760