In - Vivo Conversion of Astrocytes to Neuroblasts in the Injured Spinal Cord
Summary (3 min read)
Introduction
- It is shown that the SRY (sex-determining region Y-box 2), also known as Sox2, is a transcription factor that is essential for maintaining self-renewal, or pluripotency, of undifferentiated embryonic stem cells.
- Astrocytes due to nearness to radial glial cells identity, their high quantity, and the potential to proliferate in the central nervous system, astrocytes could undertake therapeutic interventional approaches including reprogramming.
- The concept of direct reprogramming is a process in which one mature somatic cell transforms into another mature somatic cell without undergoing an intermediate pluripotent state or progenitor cell type(Ghasemi- Kasman et al. 2015) .
Ethical issue and study design
- In the current study, 54 adult male Wister rats (weight: 270-300g) were purchased from the animal laboratory and maintained according to the guide line of ethics committee of Tabriz University of Medical Sciences (registered number 95/5-10/7).
- All animals were housed in a standard condition under a 12h light/dark schedule with enough food and water.
- In all groups, laminectomy was performed at the T9-T10 vertebral level in the dorsal surface of the spinal cord using the In nite Horizons Impactor with an impact force of 150 (moderate SCI) kdyn (1 dyn=1g⋅cm/s 2=10-5kg⋅m/s 2=10-5 N) by impactor device.
- Amniotic uid extraction carried out under supervision the gynecologist with using a 22G Needle.
- In the cultivation period, the medium was changed twice a week, and in the 90% con uence, the cells trypsinized with trypsin-EDTA [0.25%] and centrifuged, and nally, cells pellet re-seeded in DMEM-low glucose media with 15% FBS, 1% penicillin/streptomycin and10 ng/mL bFGF in the optimized condition.
Preparation of CM
- The MSCs at the 3rd-5rd passage and 90% con uent, were trypsinized and washed with PBS three times, and re-fed with DMEM-low glucose culture medium in serum-free condition for 48 h.
- Finally, CM was sterilized through 0.22 µm lters and concentrated by freeze-drying processes and was stored at -80 ℃ until use.
Spinal Cord Surgery
- After deep anesthesia, an adequate level of anesthesia was determined by checking withdrawal to painful stimuli applied to the hind limb.
- The rats were shifted to the surgical location and via the mask received an iso urane vapor inhalation (3-5%) and oxygen (0.8-1 L/min) to the end of surgical procedure.
- To prevent infection following SCI, cipro oxacin (350 ml units/days) was injected via IP for one week.
- After SCI, the bladder sac was discharged manually twice a day for one week.
Transplantation of hAF-MSCs
- The next, to examine the effect of the hAF-MSCs on endogenous expression of neuroblasts and astrocytes, the MSCs were detached and harvested using 0.25% Trypsin-EDTA.
- Prior to the transplantation, the number of cells was estimated by counting in a neobar lam and washed by PBS three times.
- Following SCI, 5×105 cells per 5µl PBS were transplanted to the proximal, central, and distal parts of the lesion site using a capillary glass needle through a Hamilton syringe.
- For immunosuppression, the rats received cyclosporine (1 mg/100 g body weight) two days before cell transplantation until the end of the experiment (Springer et al. 2018 ).
Injection of the exogenous human astrocytes
- In the next series of experiments, the authors decided to investigate the effect of the MSCs and their CM on astrocyte reprogramming and converting the human astrocytes to neuroblasts.
- To this end, the human astrocytes (Cell line 1321N1) were injected focally into the lesion site of the spinal cord concomitantly with transplantation of MSCs and infusion of CM.
Tissue preparation and immuno uorescence staining
- For immuno uorescence examinations, animals were sacri ced two weeks after SCI by ketamine (100mg/kg) and xylazine (5mg/kg) overdose.
- The rats were transcardially perfused with normal saline (NaCl 9%) and 4% paraformaldehyde, respectively.
- The animal's spinal cord was carefully removed and post-xed overnight with 4% paraformaldehyde solution.
Statistical analysis
- One-way analysis of variance and post hoc.
- Tukey tests were performed to detect the statistically signi cant difference between groups.
- P<0.05 was considered as statistically signi cant.
- All the statistics presented in the article were analyzed and drawn using Graph Pad Prism (version 6.01; Graph Pad Software, CA, USA).
MSCs secreted the SOX2 protein in the CM
- This analysis showed that the Sox2 protein was observed in the 32 folds concentrated CM.
- The results from this panel showed that the MSCs after two weeks of transplantation promoted the level of DCX expression and suppressed the rate of GFAP marker.
- In general, these data indicate the MSCs through juxtacrine and paracrine pathways could promote the neuroblasts and induce neurogenesis, as well as, diminish the astrocytes in the SCI and nally suppress gliosis and glial scar formation.
- MSCs and CM could increase the number of neuroblasts relative to the (SCI +Astrocytes + DMEM) group (p<.001, p<.05) respectively .
- Moreover, presented data showed that MSCs in comparison to CM could raise the functional recovery score more than the CM(p<.05) .
Discussion
- Astrocytes in large quantities have been distributed throughout the CNS and provide essential factors and desire microenvironment for optimal neural tissue structure and function in a healthy condition (Sofroniew and Vinters 2010) .
- Regardless of these ndings, astrocyte-derived neurons have not identi ed around the injured area in both the brain and spinal cord (Ohori et al.
- Therefore, nding new and effective therapeutic strategies including reprogramming and neurogenesis promotion is a challenging and debate subject in the regenerative medicine eld.
- According to previous evidence, transcription factor Sox2 could be su cient to converting endogenous differentiated astrocytes into neuroblasts and also mature neurons in the adult spinal cord with different severity of the injury (Su et al. 2014a ).
- In conclusion, the results of the present research point out the protective and regenerative potential of MSCs in the SCI through reprogramming astrocytes to neuroblasts by mediating juxtacrine activity and paracrine effects.
Declarations
- Author Contributions M.PA and P.SH designed and supervised the whole study.
- M.K prepared and wrote the manuscript and analyzed data.
- M.A performed the experiments and involved in data acquisition.
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References
285 citations
Additional excerpts
...Several previous studies have used CM in various disorders like skin wounds (Chen et al. 2008), CNS (Teixeira et al. 2014), hepatic transplant (Du et al. 2013), acute lung injury (Ionescu et al. 2012), and chronic kidney diseases (van Koppen et al. 2012)....
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271 citations
"In - Vivo Conversion of Astrocytes ..." refers background in this paper
...Sox2 which is available in hAF-MSCs-CM is the essential factor for the transform endogenous spinal astrocytes to neuroblasts (Su et al. 2014b; Zhang and Cui 2014)....
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...Sox2 has a critical role in the maintenance of embryonic and neural stem cells and also it is crucial for directing neural differentiation (Zhang and Cui 2014; Mercurio et al. 2019; Rodriguez-Jimenez et al. 2016)....
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206 citations
"In - Vivo Conversion of Astrocytes ..." refers background in this paper
...Li H, Chen G (2016) In vivo reprogramming for CNS repair: regenerating neurons from endogenous glial cells....
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...Current Drug Targets-CNS & Neurological Disorders 4 (1):41-50 32....
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...In response to any CNS damage, they are activated, proliferated, migrated to the lesion site, and participate in glial scar formation (Sofroniew 2009; Yiu and He 2006; Buffo et al. 2008)....
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...Sun X, Tan Z, Huang X, Cheng X, Yuan Y, Qin S, Wang D, Hu X, Gu Y, Qian W-J (2019) Direct neuronal reprogramming of olfactory ensheathing cells for CNS repair....
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...A plethora of studies has shown that the activated astrocytes in CNS injuries could be isolated and cultivated in vitro cultivation and produce neurospheres with the capability of a generation of neurons, astrocytes, and oligodendrocytes....
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200 citations
"In - Vivo Conversion of Astrocytes ..." refers background in this paper
...Regardless of these ndings, astrocyte-derived neurons have not identi ed around the injured area in both the brain and spinal cord (Ohori et al. 2006; Buffo et al. 2008; Buffo et al. 2005)....
[...]
196 citations
"In - Vivo Conversion of Astrocytes ..." refers background in this paper
...MSCs during cell culture can secrete paracrine factors in the form of conditioned medium (CM) (Osugi et al. 2012; Cantinieaux et al. 2013)....
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...(Cantinieaux et al. 2013; Pawitan 2014)....
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