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In - Vivo
Conversion of Astrocytes to Neuroblasts in
the Injured Spinal Cord
Maryam Lale Ataei
Tabriz Medical University: Tabriz University of Medical Sciences
Mohammad Karimipour ( karimipourm@tbzmed.ac.ir )
Tabriz University of Medical Sciences https://orcid.org/0000-0002-6975-4308
Parviz Shahabi
Tabriz Medical University: Tabriz University of Medical Sciences
Hamid Soltani-Zangbar
Tabriz Medical University: Tabriz University of Medical Sciences
Maryam Pashaiasl
Tabriz Medical University: Tabriz University of Medical Sciences
Research Article
Keywords: Spinal cord injury, MSCs, Astrocyte, Neuroblast, Reprogramming
Posted Date: April 26th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-362556/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Direct astrocyte reprogramming to neural progenitor cells and promotion of neurogenesis is considered
as an alternative approach to replace the lost neurons in the spinal cord injury(SCI). Herein, we used the
human amniotic uid mesenchymal stem cells (hAF-MSCs) and their conditioned medium (CM), to
investigating their ability to reprogramming astrocytes to neuroblasts following SCI. 54 adult rats were
randomly divided into 9 groups (n = 6), included: Control, SCI, (SCI + DMEM), (SCI + CM), (SCI + MSCs),
(SCI + Astrocyte), (SCI + Astrocyte + DMEM), (SCI + Astrocyte + CM) and SCI + Astrocyte + MSCs).
Following laminectomy and SCI induction, DMEM, CM, MSCs and Astrocytes were injected. Wester-blot
was performed to explore the levels of the Sox2 protein in the MSCs-CM. The immunouorescence
staining against DCX and GFAP was done. Finally, Basso-Beattie-Brenham (BBB) locomotor test was
conducted to assess the neurological outcomes. Our results showed that the MSCs through juxtacrine
and paracrine mechanisms induced the promotion of the endogenous neuroblasts and the decline of
astrocytes. Moreover, in the present research, MSCs and CM could convert the transplanted human
astrocytes to neuroblasts in the spinal cord injury. Taken together, our data indicate the MSCs via
juxtacrine and paracrine pathways could direct the spinal cord endogenous neural stem cells(NSCs) to
the neuroblasts lineage rather than astrocytes as well as induce reprogramming. Ultimately, MSCs could
reverse the neurobehavioral decit in the SCI. The striking output in our study was the capability of the
MSCs in the reprogramming of the astrocytes to neuroblasts via juxtacrine and paracrine pathways in the
In-vivo
condition.
Introduction
Spinal cord injury (SCI) is damage to the spinal cord and a complex neurological condition that resulted
in irreversible neuronal loss, glial scar formation, axonal injury, disruption of the myelin sheath and neural
tracts and long-lasting disability and in some cases leads to permanent functional decits in
mammals(Su et al. 2014b; Higuchi et al. 2019; Abolhasanpour et al. 2019). Following SCI, astrocytes
proliferate and migrate into the surrounding milieu and generate glial scar and undesirable
microenvironment (Wang et al. 2016). The research demonstrated that the astrocytes and NG2 (Pre-
oligodrocytes), as a result of reprogramming could convert to neuroblast and neurons in the spinal cord
and brain (Wang et al. 2016). 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. 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). Sox2 can mediated reprogramming of astrocytes to DCX +
neuroblasts and in this context, Sox2 can help to treat SCI by converting glial cells to neurons nally
mature neurons (Wang et al. 2016; Rodriguez-Jimenez et al. 2016). Doublecortin (DCX) is a microtubule-
associated protein expressed by neuronal precursor cells and immature neurons in embryonic and adult
cortical structures and considered as a marker of developing neural progenitor cells (Karimipour et al.
2019; Nasrolahi et al. 2019). Astrocytes due to nearness to radial glial cells identity, their high quantity,
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and the potential to proliferate in the central nervous system, astrocytes could undertake therapeutic
interventional approaches including reprogramming. (Sun et al. 2019). Recent advancements in the eld
of direct in vivo reprogramming showed the generation of functional neurons from reactive glial cells in
the repair of the brain (Li and Chen 2016; Guo et al. 2014). 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).
Transplantation stem cells including MSCs and olfactory ensheathing cells (OECs) in SCI could lead to
increase of neuroprotective molecules and reprogramming of astrocytes into neuroblasts which could
survive and develop into mature neurons (Sabapathy et al. 2017; Sun et al. 2019). In this context, MSCs
by secretion of the essential materials for neuroprotection such as growth factors, cytokines, and
neurotrophic factors, could induce the replacement of lost neuronal cells, remyelination of axons,
angiogenesis, and decline of inammatory responses(Oh and Jeon 2016; Shahrezaie et al. 2017). MSCs
could harvest from different sources including adipose derived tissue, bone marrow, Umbilical Cord
Wharton's Jelly, amniotic membrane and amniotic uid mesenchymal stem cells (hAF-MSCs) and so on
(Gabel et al. 2017; Oh and Jeon 2016). In spite of the plethora of studies and experiments regarding the
evaluation of the functional ecacy of the MSCs in the SCI, the election of the effective source of the
MSCs is one of the challenging issues in the scientic community (Gabel et al. 2017). But recent studies,
suggest that (hAF-MSCs) could account into consideration in the SCI because of the amnion origin from
the epiblast layer as a pluripotent and undifferentiated structure in the embryo(De Coppi et al. 2007;
Antonucci et al. 2014). These cells routinely gained during amniocentesis which is done in the second
trimester and express embryonic and pluripotency markers for instance Oct4, Nanog and Sox2 (Aziz et al.
2019; Gholizadeh-Ghaleh Aziz et al. 2019; Dziadosz et al. 2016; Maraldi et al. 2014; Saito et al. 2012).
Amniotic uid derived- MSCs relative to other sources have high proliferative and regenerative potential
as well as they maintain pluripotency features, and remain without alteration after continuous passages
(Maraldi et al. 2014; Aziz et al. 2016). MSCs during cell culture can secrete paracrine factors in the form
of conditioned medium (CM) (Osugi et al. 2012; Cantinieaux et al. 2013). CM contains metabolites,
growth factors, extracellular matrix proteins, cytokines and anti-inammatory agents which are secreted
into the medium by the cultured cells. (Cantinieaux et al. 2013; Pawitan 2014). 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). In this Perspective, we investigated the eciency of the hAF-MSCs
and their CM in the reprogramming of the astrocytes to neuroblasts through juxtacrine and paracrine
mechanisms as well as functional behavior following the SCI.
Materials And Methods
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. The rats were randomly divided in to 9 groups (six rats
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per group), these nine groups included:Control,SCI, (SCI + DMEM, IP), (SCI + CM, IP), (SCI + MSCs,
Focally), (SCI + Astrocyte), (SCI + Astrocyte+ DMEM, IP), (SCI + Astrocyte+ CM, IP) and SCI + Astrocyte+
MSCs). In all groups, laminectomy was performed at the T9–T10 vertebral level in the dorsal surface of
the spinal cord using the Innite 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.
hAF-MSCs isolation, cultivation and characterization
Briey, isolation of hAF-MSCs was done undergoing amniocentesis for the routine karyotype screening of
about 5 ml of amniotic uid samples from eight mothers in Al-Zahra hospital (Tabriz, Iran). Prior to the
amniocentesis, patients written informed consent for donating amniotic uid samples voluntarily for this
research. Amniotic uid extraction carried out under supervision the gynecologist with using a 22G
Needle. After sending the samples to the desired laboratory, samples were centrifuged at 450 g for 10
minutes, next the settled pellet was washed twice by PBS (Gibco; Thermo Fisher Scientic, Darmstadt,
Germany). Then, the cells transferred in 6 well plates with AmnioMAX II Complete Medium (Gibco, cat#
11269) for 1-2 weeks in condition 37°C and 5% CO2. In the cultivation period, the medium was changed
twice a week, and in the 90% conuence, the cells trypsinized with trypsin-EDTA [0.25%] (Gibco) 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. The phenotype prole of the hAF-
MSCs was examined by ow cytometry analysis. For this purpose, the cells at passage 3-5 were
trypsinzed and two times washed with PBS and centrifuge at 1500 RPM for 3 min, then, the cell pellets
were stained with antibodies including CD 105 (Catalog No. 1p-298-To25 Exbio), CD 73 (Catalog No.
561260 BD Biosciences) antibodies as mesenchymal stem cell markers and CD 45 (Catalog No. 1F-222-
T025 Exbio), CD 14 (Catalog No. 12-0149-42 eBioscience) as hematopoietic stem cell markers) with
dilution 1/30 in the PBS for 20 min on ice.
Preparation of CM
The MSCs at the 3rd-5rd passage and 90% conuent, were trypsinized and washed with PBS three times,
and re‐fed with DMEM-low glucose culture medium in serum‐free condition for 48 h. Then, CM was
harvested and centrifuged at 450g for 10 min up to eliminate free-oating cells. Finally, CM was sterilized
through 0.22 µm lters and concentrated by freeze-drying processes and was stored at -80 ℃ until use.
Western blot
CM was collected from MSCs culture and was sterilized by 0.22 mm lters, then CM was concentrated 2,
4, 8, 16 and 32 folds by freeze dryer device. Sox2 (Sex determining region Y-box 2) secreted by HAF-MSCs
into MSCs-CM, was measured using western blot analysis.
Human astrocyte culture
Human astrocytes (line 1321N1) (Ghasemi-Kasman et al. 2015) were purchased from Pasteur Institute of
Iran and cultured in DMEM low glucose medium with 10% FBS and 1% penicillin/streptomycin. This
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medium was changed twice in a week.
Spinal Cord Surgery
The rats were anesthetized by inhalation of 5% isourane and oxygen (1 L/min) in a closed space. 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 isourane vapor inhalation (3-5%) and oxygen (0.8-1 L/min) to the end of surgical procedure. Briey,
animals under anesthesia conditions, their back was shaved and in the midline, the skin s incision was
performed around 2cm, and in order to laminectomy, Paravertebral muscles were cut up on the T9-T11
spinal processes, and with the dental drill, a hole of 1.5 mm diameter was made in the vertebral arch of
T10 as far as dura mater could be seen. Then, using the Horizons Impactor, animals received a force of
150-kilodyne (moderate SCI) on the targeted spinal cord segments, subsequently, the muscles and skin
were closed. Also for bilateral injury, transverse process of the rats throughout the surgery and injury xed
by a clamp. To prevent infection following SCI, ciprooxacin (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.
Infusion of MSCs- CM
In order to explore the effect of the CM on the rate expression of the endogenous neuroblasts and
astrocytes, 500 µl of CM, following SCI was infused through intraperitoneal (IP) per day for 7 days.
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, we 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 immunouorescence staining
For immunouorescence examinations, animals were sacriced two weeks after SCI by ketamine
(100mg/kg) and xylazine (5mg/kg) overdose. The rats were transcardially perfused with normal saline