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. 2018 Aug 17;19(8):2434.
doi: 10.3390/ijms19082434.

Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo

Soomi Jang  1 , Young-Hoon Kang  2   3 , Imran Ullah  4 , Sharath Belame Shivakumar  5 , Gyu-Jin Rho  6 , Yeong-Cheol Cho  7 , Iel-Yong Sung  8 , Bong-Wook Park  9   10
Affiliations

Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo

Soomi Jang et al. Int J Mol Sci. .

Abstract

The reduction of choline acetyltransferase, caused by the loss of cholinergic neurons, leads to the absence of acetylcholine (Ach), which is related to motor nerve degeneration. The aims of the present study were to evaluate the in vitro cholinergic nerve differentiation potential of mesenchymal stem cells from cryopreserved human dental pulp (hDPSCs-cryo) and to analyze the scale of in vivo motor nerve regeneration. The hDPSCs-cryo were isolated and cultured from cryopreserved dental pulp tissues, and thereafter differentiated into cholinergic neurons using tricyclodecane-9-yl-xanthogenate (D609). Differentiated cholinergic neurons (DF-chN) were transplanted into rats to address sciatic nerve defects, and the scale of in vivo motor nerve regeneration was analyzed. During in vitro differentiation, the cells showed neuron-like morphological changes including axonal fibers and neuron body development, and revealed high expression of cholinergic neuron-specific markers at both the messenger RNA (mRNA) and protein levels. Importantly, DF-chN showed significant Ach secretion ability. At eight weeks after DF-chN transplantation in rats with sciatic nerve defects, notably increased behavioral activities were detected with an open-field test, with enhanced low-affinity nerve growth factor receptor (p75NGFR) expression detected using immunohistochemistry. These results demonstrate that stem cells from cryopreserved dental pulp can successfully differentiate into cholinergic neurons in vitro and enhance motor nerve regeneration when transplanted in vivo. Additionally, this study suggests that long-term preservation of dental pulp tissue is worthwhile for use as an autologous cell resource in the field of nerve regeneration, including cholinergic nerves.

Keywords: cell transplantation; cholinergic nerve differentiation; dental pulp stem cells; tissue cryopreservation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Morphologies, culture characteristics, and cell proliferation rates of cryopreserved human dental pulp tissues harvested from extracted wisdom teeth (hDPSCs-cryo). (A) Cell morphologies of hDPSCs-cryo at passage 0 (P0) and passage 3 (P3) showing homogeneous plate adherent fibroblast-like morphology (scale bar = 100 µm); (B) Cell proliferation rate by population doubling time (PDT) of hDPSCs-cryo produced normal cell proliferation curvatures. Data represent the mean ± SD of three independent experiments; (C) Cell cycle analysis of hDPSCs-cryo showed normal DNA content in the gap 0/1 (G0/G1), synthesis (S), or gap 2/mitotic (G2/M) phases of the cell cycle; (D) Western blot analysis revealed positive protein expression of the pluripotent markers, octamer-binding transcription factor 4 (Oct4), Nanog, and sex determining region Y-box 2 (Sox2); and (E) The relative quantification of pluripotent marker expressions (D) to the control protein (β-actin) revealed relatively higher expression of Nanog over Oct4 or Sox2. Data represent the mean ± SD of three independent experiments.
Figure 2
Figure 2
Characterization of hDPSCs-cryo at passage 3. (A,B) Fluorescence-activated cell sorting (FACS) analysis for hematopoietic and mesenchymal stem cell (MSC) markers revealed high MSC-marker expression (cluster of differentiation (CD)29, CD73, and CD90), whereas hematopoietic markers (CD34 and CD45) were almost negatively expressed; (C) hDPSCs-cryo showed successful in vitro differentiation potential to mesenchymal lineage, as confirmed by lineage specific staining (Oil red O for adipocytes, Alizarin red and von Kossa for osteocytes, and Safranin O and Alcian blue for chondrocytes; scale bar = 100 µm); and (D) The messenger RNA (mRNA) levels of lineage-specific genes were analyzed using quantitative real-time PCR (RT-qPCR) with the 2−ΔΔCt method using tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) for normalization. Expression levels are expressed as fold change relative to the control (undifferentiated hDPSCs-cryo) [peroxisome proliferator-activated receptor gamma-2 (PPARγ2), lipoprotein lipase (LPL), and fatty-acid binding protein 4 (FABP4), adipocyte-specific; bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), and osteonectin (ON), osteocyte-specific; and sex determining region Y-box 9 (SOX9), cartilage-specific proteoglycan core protein (AGGRECAN); and type II collagen (COLLAGEN II); chondrocyte-specific]. Data represent the mean ± SD of three independent experiments. * denotes significant differences, p < 0.05.
Figure 3
Figure 3
Morphological changes during cholinergic neuronal differentiation of hDPSCs-cryo and expression levels of cholinergic neuron-specific markers. (A) Morphology of hDPSCs-cryo (day 0) changed to neuron-like cells, possessing neuronal body and axonal fibers, after the induction time passed (day 2 and day 3) (scale bar = 50 µm); (B) Differentiated cholinergic neurons (DF-chN) at day 3 showed increased mRNA levels of cholinergic-specific genes, choline acetyltransferase (ChAT), homeobox HB9 (HB9), and insulin gene enhancer protein ISL-1 (ISL1), using RT-qPCR. Data represent the mean ± SD of three independent experiments (* denotes significant differences, p < 0.05); and (C) Cholinergic marker protein expression using Western blot analysis in both differentiated neurons (DF-chN) and undifferentiated control (hDPSCs-cryo). DF-chN after tricyclodecane-9-yl-xanthogenate (D609) treatment in hDPSC-cryo showed increased expression levels of cholinergic-specific proteins, ChAT, HB9, and ISL1, whereas the expression of these marker proteins in undifferentiated hDPSCs-cryo was undetectable.
Figure 4
Figure 4
Immunocytochemical analysis of DF-chN (A) and undifferentiated hDPSCs-cryo (B) for cholinergic-specific proteins. Similar to the Western blot analysis, DF-chN with D609 treatment revealed strong expression of cholinergic-specific proteins, ChAT, HB9, and ISL1, whereas the same proteins were not expressed in undifferentiated hDPSCs-cryo (Scale bar = 50 µm).
Figure 5
Figure 5
Analysis of acetylcholine (Ach) levels in spent media using a biochemical fluorescent assay. The culture media of DF-chN showed increased Ach levels compared to undifferentiated hDPSCs-cryo, indicating DF-chN could synthesize Ach (mean ± SD of three different experiments; * denotes significant differences, p < 0.05).
Figure 6
Figure 6
Schematic images and immunohistochemical analysis of in vivo cell transplantation in experimental rats with nerve defects. (A) The sciatic nerve was exposed on the thigh of the experimental animal; (B) The sciatic nerve was resected and a 5-mm nerve gap developed; (C) After tubulization with biodegradable collagen bovine dura mater (Lyoplant®) in the nerve gap, a total of 1 × 106 cells (DF-chN) were transplanted into the collagen tube with a 0.1-mL fibrin glue scaffold (Greenplast®). The tube was then sealed with sutures; (D) Photograph of the experimental nerve at eight weeks after DF-chN transplantation. In the cell transplantation site, the regeneration of nerve defect was detected with continuity of the nerve gap; (E–I) Histological and immunohistochemical analysis of low-affinity nerve growth factor receptor (p75NGFR) in regenerated motor nerve fibers at eight weeks post cell transplantation. (E) In non-cell-transplanted control specimens, regenerated nerve fibers were not detected in abundance and p75NGFR expression was weak. (F) In the DF-chN transplanted group, significantly increased regenerating nerve fibers were detected in the nerve gap (arrows). The asterisk (*) indicates the proximal end of the nerve gap. (G,H) High magnification of generated nerve fibers at the DF-chN graft site. High expression of p75NGFR was detected in newly generated nerve fibers, especially in the perineurium and endoneurium of regenerating nerve fibers. (I) Toluidine blue staining of regenerating nerve fibers showed blue-colored positive staining of the myelin sheath, which was detected in abundance in the outer layer of newly generated axonal fibers (Scale bar = 100 µm).
Figure 7
Figure 7
PKH26 expression and histological analysis of the regenerated nerve fibers at eight weeks after cell transplantation. (A) In the nerve connection site, the regenerated nerve (defect site) was connected to the cutting end of proximal portion of sciatic nerve (sciatic nerve), black arrows indicating the connection portion. In the same specimen, the fluorescence intensity of pre-transplanted cell-labeling dye PKH26 was remarkably detected in the DF-chN transplanted site (white arrows), demonstrating that the transplanted DF-chN have excellent in vivo adaptability and proliferation abilities. Interestingly, some transplanted cells were also detected in the original sciatic nerve portion (black arrows in Merge), indicating the transplanted DF-chN could move to the original sciatic nerve portion and communicate with the host cells; and (B) Histological features of proximal, middle, and distal portions of the nerve defect site. In the proximal portion, black arrows indicate the connection site between the cell-transplanted site and the nerve cutting end. Abundant newly generated blood vessels were detected in this portion (white arrows). In the middle and distal portion of the nerve defect, homogeneous newly generated nerve fibers were evenly detected (Scale bar = 100 µm).
Figure 8
Figure 8
Behavioral analysis of the experimental rats at eight weeks post cell transplantation. The walking ability of experimental rats was evaluated using the open-field test. Over a 10-min period, the velocity (A), distance moved (B), and number of wall climbs (C) were investigated; and (D) Representative walking trace of rats at eight weeks post experimental operation. Control, non-cell transplanted negative control; Cell Trans, DF-chN-transplanted group; Sham OP, Sham operation (OP) group. Data represent the mean ± SD of three independent experiments (different letters denote statistically significant differences between groups, p < 0.05).
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