347

ORIGINAL
Effective in vitro differentiation of adipose-derived stem cells
into Schwann-like cells with folic acid supplementation
Shuhai Chen, Tetsuya Ikemoto, Takuya Tokunaga, Shohei Okikawa, Katsuki Miyazaki, Kazunori Tokuda,
Shinichiro Yamada, Yu Saito, Satoru Imura, Yuji Morine, and Mitsuo Shimada
Department of Digestive and Transplant Surgery, Tokushima University, Tokushima, Japan

Abstract : Peripheral nerve injury (PNI) after pelvic surgery is a common issue with a significant impact on patients. Autologous nerve grafting is the gold standard treatment for PNI, but this technique cannot be applied
to fine nerve fibers in the pelvis. Schwann-like cell (SLC) differentiation is a novel therapeutic strategy for thisclinical condition. However, the efficiency of SLC differentiation remains unsatisfactory. We modified an SLC
differentiation protocol using adipose-derived stem cells (ADSCs) and folic acid. Morphology, gene expression
and secretion of neurotrophic factors were examined to assess the differentiation quality and phenotypic characteristics. Our new modified protocol effectively induced a Schwann cell (SC) phenotype in ADSCs as assessedby morphology and expression of SC markers [S100 calcium-binding protein B (S100B), P < 0.01 ; p75 neurotrophic
receptor (p75NTR), P < 0.05]. SLCs produced by the new protocol displayed a repair phenotype with decreased
expression of ERBB2 and early growth response protein 2 (EGR2) / KROX20 (P < 0.01). Furthermore, our new protocol enhanced both mRNA expression and secretion of nerve growth factors by SLCs (P < 0.01). This protocolenhanced the SC characteristics and functions of ADSC-derived SLCs. This promising protocol requires further
research and may contribute to SC-based nerve regeneration. J. Med. Invest. 68 : 347-353, August, 2021
Keywords : Adipose-derived stem cell, Schwann-like cells, Folic acid, Nerve growth factor, Regenerative medicine

INTRODUCTION
Peripheral nerve injury (PNI) is a common issue that affects
more than 1 million people every year worldwide and causesa significant degree of pain in the affected individuals (1). Autologous nerve transplantation is currently the best method fortreating PNI, but its application is limited owing to the scarcity
of donors and the risk of neuroma formation and other complications due to a second operation (2). Moreover, PNI after pelvic
surgery needs to be addressed to improve patients’ quality oflife (3). Nerve transplantation is one option ; however, injured
nerve fibers are generally fine and complex. Therefore, anotherstrategy is required.
Cell transplantation is another option that has progressively
gained popularity. Schwann cells (SCs) contribute to the removalof damaged myelin, secretion of nerve growth factors (NGFs)
and the formation of new myelin. These properties are beneficial
for the regeneration of peripheral nerves (4, 5). However, owing
to limitations in SC separation, purification and proliferation
technology, there is an urgent requirement for abundant alternative cells with lower immunogenicity. Regenerative medicine
has recently been applied to solve these issues (6). Adipose-derived stem cells (ADSCs) have been focused on because of theirmultipotency and ease of harvest. We have investigated ADSC
multipotency and reported scientific evidence of insulin producing cells (IPCs) for clinical applications (7-11). Thus, we hypothesized that ADSCs can also self-renew and differentiate into
ectodermal lineage cells, including Schwann-like cells (SLCs).
The most commonly used differentiation protocol for producing
Received for publication March 1, 2021 ; accepted August 19, 2021.
Address correspondence and reprint requests to Tetsuya Ikemoto,
MD, PhD, FACS, Department of Digestive and Transplant Surgery,
Tokushima University, 3-18-15 Kuramoto, Tokushima, 770-8503,
Japan and Fax : +81-88-631-9698.

SLCs from ADSCs has been described by Kingham et al. (12).
However, this method is still not ideal, considering the scope forfurther improvement with regard to the secretion of neurotrophic factors by differentiated SLCs. Thus, it is imperative to findmethods that will ensure the optimal performance of SLCs.
Folic acid (FA) is an inexpensive and safe substance withregulatory properties in various cells in the nervous system. It
can promote chemotactic responses in glial cells, induce neurotrophin release and stimulate neuronal differentiation (13). FA
can effectively promote growth, repair and recovery of damaged
adult central nervous system (CNS) (14), provide neuroprotection and promote nerve regeneration after ischemic injury (15).
The FA / Dihydrofolate reductase (DHFR) / AMP-activated protein kinase (AMPK)α axis improves survival and induces myelination of oligodendrocytes, and thus, enhances the maturity ofoligodendrocytes in vivo and in vitro (16). FA supplementation has
been shown to be effective in PNI treatment (17). A recent study
on a rat PNI model has demonstrated that FA promoted proliferation and migration of SCs and secretion of NGF to improve
the repair of peripheral nerve damage (18). SCs transform into a
repair phenotype at the site of the injured nerve to promote nerve
regeneration (6). Then, NGFs promote autophagy of SCs throughthe p75 neurotrophin receptor (p75NTR) / AMPK / mechanistic
target of rapamycin (mTOR) signaling pathway to accelerate
removal and phagocytosis of myelin debris and promote theregeneration of axons and myelin in the early stages of PNI (19).
The close relationship between FA and NGFs indicates they playa crucial role in SC-mediated nerve regeneration. However, there
is no research on the application of FA in the differentiation process of SLC from stem cells. And the appropriate concentrationof FA also needs to be investigated.
Our new modified protocol effectively induced an SC phenotype in ADSCs as assessed by morphology and expression of SC
markers. Furthermore, new generated SLCs display a repair
phenotype as decreases of Erb-B2 receptor tyrosine kinase 2(ERBB2) and early growth response 2 (EGR2) gene expression,

The Journal of Medical Investigation Vol. 68 2021

348

S. Chen, et al. Schwann-like cells from ADSCs using folic acidand show enhanced secretion capacity of NGFs. This promising
new protocol requires further research for SC-mediated nerve
regeneration.

MATERIALS AND METHODS

Cell Culturing and Differentiation Protocols
Human ADSCs were purchased from Invitrogen (Grand
Island, NY, USA) and cultured in ADSC basal medium withadded growth supplement (Gibco, Carlsbad, CA, USA) and
GlutaMAX-I (Gibco). Primary human SCs (ScienCell ResearchLaboratories, Carlsbad, CA, USA) were cultured in 6-wellflat-bottom plates precoated with poly-L-lysine (2 μg / cm2 , P4707,
Sigma-Aldrich) and maintained in complete SC medium supplemented with 5% fetal bovine serum (FBS), 1% SC growth
supplement cocktail and 1% penicillin / streptomycin (ScienCell
Research Laboratories) following the manufacturer’s instructions. To produce SCs (50 μg / ml) group, 50 μg / ml of FA wasadded into the growth medium to stimulate SCs for 3 days.
For inducing conventional SLCs differentiation, a three-step
protocol reported by Kingham et al. (12) was used. Briefly,
ADSCs were seeded in collagen I-coated 6-well plates (4810010, IWAKI, Tokyo, Japan), cultured in α-modified Eagle’s
medium (α-MEM, Gibco) without FBS (Gibco) and treated with
1 mM β-mercaptoethanol (β-ME, 133-14571, Fujifilm Wako
Pure Chemical Corporation, Osaka, Japan) for 24 h. Next,
α-MEM with 10% FBS, 35 ng / ml all-trans-retinoic acid (ATRA,
3027949, PeproTech Ltd., Cranbury, NJ, USA) were followed totreat cells for 72 h. Then, α-MEM with 10% FBS and a mixtureof growth factors including 252 ng / ml glial growth factor-2(GGF-2, 90255-B, BPS Bioscience, Inc., San Diego, CA, USA), 10
ng / ml basic fibroblast growth factor (bFGF, 100-18B, PeproTechLtd.), 5 ng / ml platelet-derived growth factor (PDGF, 100-14B,
PeproTech Ltd.) and 14 μM Forskolin (F3917, Sigma-Aldrich)were used to culture cells for 14 days with a medium change
for every 3 days. For our new protocol, 50 μg / ml FA (F8758-5G,
Sigma-Aldrich, St. Louis, MO, USA) was added as supplementation in the step 3 as the schematic representation shown in Fig.
1. We called these SLCs as SLCs (FA). In addition, for checking
the effects of different concertation of FA on the differentiation ofSLCs, 10 μg / ml and 200 μg / ml FA were also used in the step 3of differentiation protocol and these SLCs were named as SLCs
(FA, 10 μg / ml) and SLCs (FA, 200 μg / ml), respectively.

Morphological Analysis
Cells were seeded in a Nunc Lab-Tek II Chamber Slide System (154526PK, Thermo Fisher Scientific, Waltham, MA, USA)and stained with hematoxylin and eosin (H&E). Groups of cells
were compared under an optical microscope.

Quantitative Real-Time Reverse Transcription Polymerase Chain
Reaction (qRT-PCR)
An RNeasy Mini Kit (Qiagen, Hilden, Germany) and a reverse
transcription kit (Applied Biosystems, Thermo Fisher Scientific)
were used to extract total RNA, which was reverse transcribedto obtain cDNA. qRT-PCR analyses were performed using a StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo
Fisher Scientific). The primers (TaqMan gene expression assays,
Thermo Fisher Scientific) used were : S100B (Hs00902901_
m1), p75NTR (Hs00609976_m1), ERBB2 (Hs01001580_m1),
EGR2 / KROX20 (Hs00166165_m1), NGF (Hs00171458_m1)
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)(Hs99999905_m1). Results are presented as relative mRNA
expression normalized to GAPDH as an internal control.

Immunofluorescence Staining
Indicated cells were digested by trypsin and seeded for
2×104 / well in a Nunc Lab-Tek II Chamber Slide System untiladhesion. After being washed twice with PBS, 4% paraformaldehyde (163-20145, FUJIFILM) was used to fix the cells
at 4°C for 30 min. Next, incubated the slides with 0.1% Triton
X-100 (HFH10, Thermo Fisher Scientific, Inc.) for 2 min at room
temperature for permeabilization and followed with 3% bovine
serum albumin (BSA) diluted from MACS BSA Stock Solution(130-091-376, Miltenyibiotec, Bergisch Gladbach, Germany) in
PBS for 1 h. all slides were incubated. Then, the slides were incubated with an Anti-S100 beta antibody (ab52642, 1 : 100, Abcam,
Cambridge, UK) or Anti-p75NTR (sc-271708, 1 : 50, Santa Cruz
Biotechnology, Inc. Paso Robles, Ca, USA) at 4°C overnight. The
next day, after being washed three times with PBS, Alexa Fluor
488 conjugated secondary antibodies (A-11008, 1 : 500, Thermo
Fisher Scientific, Inc.) was used to incubate slides for S100B
and Alexa Fluor 594 conjugated secondary antibodies (A-11005,
1 : 800, Thermo Fisher Scientific, Inc.) was for p75NTR, for 1 h at
37°C in the dark. Then, slides were washed with PBS for threetimes protected from light and stained the nuclei by ProLong™
Gold Antifade Mountant with DAPI (P36931, Thermo Fisher
Scientific, Inc.) for 10 min in the dark. A fluorescence microscope(BZ-X700 KEYENCE, Tokyo, Japan) was used to observe andphotograph the slides. As for negative controls, the primary antibody was replaced with BSA.

Enzyme-Linked Immunosorbent Assay (ELISA)
Human NGF ELISA kits (BEK-2212-1P / 2P, Biosensis, Thebarton, Australia) were used to assess NGF secretion in the
supernatant of different groups of cells following the manufacturer’s instructions. Absorbance at 450 nm was measured using
a microplate reader and the concentration of NGF was calculatedusing a standard curve. Relative secretion capacity was normalized to the cell number.

Statistical Analysis
Fig 1. Schematic representation of the modified protocol fordifferentiating ADSCs into SLCs. adipose-derived stem cells,
ADSCs ; Schwann-like cells, SLCs ; β-mercaptoethanol, β-ME ; alltrans-retinoic acid, ATRA ; basic fibroblast growth factor, bFGF ; FA,
folic acid ; glial growth factor-2, GGF-2 ; platelet-derived growth factor,
PDGF.

For statistical analysis and plotting, we used Prism 7.0 software (GraphPad, San Diego, CA, USA). Data are expressedas the mean ± standard deviation. Comparisons between two
groups were evaluated using Mann-Whitney U test. Differencesamong multiple groups were tested using one way analysis ofvariance (ANOVA) followed by Tukey’s post hoc test. All experiments were performed in triplicate. All P-values are two-sided

349

The Journal of Medical Investigation Vol. 68 August 2021
and P < 0.05 was considered significant (*P < 0.05 ; **P < 0.01).

RESULTS

Improved protocol for generating SLCs
We searched the literature for methods to enhance differentiation and function of SCs or SLCs using small molecules(Table 1) (13, 18, 20-26). FA was identified as a small moleculethat may promote NGF secretion by primary SCs. Secretion ofneurotrophic factors is a crucial function of SCs for promoting
nerve regeneration, hence we hypothesized that FA would be an
inexpensive, convenient and promising candidate for enhancing
the function of SLCs. Thus, we modified our protocol by adding
50 μg / ml FA supplementation and generated SLCs (FA) (Fig. 1).

New protocol effectively induces the SC phenotype in ADSCs
Undifferentiated ADSCs grew in clusters and displayed arounded fibroblast-like shape. In contrast, SCs exhibited a classic spindle-shaped morphology with a higher aspect ratio. SLCs
(FA) became morphologically similar to SCs after 18 days of differentiation, exhibiting small and elongated spindle shapes witha bipolar or tripolar structure (Fig. 2A).

New protocol enhances the SC characteristics of generated SLCs
The generated SLCs (FA) cultured with the new protocol
displayed significant upregulation of the SC markers, S100B
and p75NTR (P < 0.05 and P < 0.01, respectively, Mann-Whitney
U test, Fig. 2B) compared with ADSCs, suggesting that the
improved protocol effectively induced the SC phenotype in SLCs
derived from ADSCs. Moreover, the immunofluorescence staining results showed that ADSCs had an extremely low expressionof S100B and a moderate expression of p75NTR, while both

high level of S100B and p75NTR were observed in SCs (Fig.
2C). SLCs (FA) generated by new protocol showed significantly
up-regulated levels of S100B and p75NTR, indicating that SLCs
(FA) were similar in phenotype to SCs (Fig. 2C). Taken together,a large proportion of ADSCs were differentiated into SLCs (FA)with SC characteristics through our modified protocol.

SLCs produced by the new protocol display a repair phenotype
ERBB2 is a subtype of Neuregulin (NRG) receptor that can
activate downstream signal transduction and facilitate themyelinating phenotype of SCs (27). EGR2 is also a marker of myelinating SCs that is downregulated in the repair phenotype andupregulated in the re-myelinating phenotype of SCs (28). Next,
we checked the effect of different concentration of FA supplementation on the phenotypic characteristics of SLCs. Compared withconventional SLCs, SLCs (FA) generated by our new protocol
with adding 50 μg / ml FA in the step 3 displayed lower mRNA
expression of both ERBB2 and EGR2 (P < 0.01, Mann-Whitney U
test, Fig. 3).

New protocol enhances the mRNA expression and the secretion of
NGF from SLCs
PCR analysis showed that SLCs (FA) generated using thenew protocol had the highest level of NGF mRNA than that in
ADSCs, primary SCs and other groups of SLCs (P < 0.01, one
way-ANOVA, Fig. 4A). Furthermore, we collected supernatantsfrom the above groups to compare NGF expression at the protein
level. The new protocol significantly increased the NGF secretion
capacity of SLCs (FA) compared with conventional SLCs (40.380± 0.803 pg / ml vs. 19.056 ± 0.580 pg / ml, P < 0.01, one way-ANOVA). In primary SCs, after stimulation with FA for 3 days, theNGF secretion also increased (12.461 ± 0.103 pg / ml vs. 49.115 ±
1.854 pg / ml, P < 0.01, one way-ANOVA).

Table 1. Summary of activators for promoting SC or SLC functions
Activator

Functions of SCs or SLCs

Feasible

Ref.

Folic acid (100 μg / ml)▪ promotes proliferation▪ promotes migration▪ promotes NGF secretion▪ promotes NT3 / NT4 / 5 secretionHigh

Kim GB, et al. (13)Kang WB, et al. (18)

Carboxymethylated chitosan(200 μg / ml)▪ promotes proliferation via the activation of Wnt / β-cateninsignaling▪ induces biosynthesis of NGF

Moderate

Tao HY, et al. (20)Lithium (30 nM)▪ promotes proliferation▪ suppresses migration

Moderate

Gu XK, et al. (21)Matrix metalloproteinase-7(10 nM)▪ promotes migration▪ no effect on proliferation▪ promotes SCs move from the proximal nerve segment andmyelin sheath formation

Moderate

Wang HK, et al. (22)Melatonin (1 nM)▪ promotes proliferation via ERK1 / 2 pathway

Moderate

Chang HM, et al. (23)

M2 receptor agonist,
Arecaidine Propargyl Ester(100 µM)▪ increases NGF production and maturation▪ decreases proliferation and migration▪ increases SC marker and myelinating marker expression▪ enhances changes in morphology

Side effect

Piovesana R, et al. (24, 25)

Immunoglobulin(20 mg / ml)▪ reduces SC proliferation rates and accelerates growth ofcellular protrusions▪ stimulates SCs maturation.
▪ boosts myelin gene expression and myelination-related
signaling pathways▪ secretes IL-18 to promote axonal growth

Immunogenicity
problemz

Tzekova N, et al. (26)Abbreviation : nerve growth factor, NGF ; neurotrophin, NT ; extracellular signal-regulated kinase, ERK ; interleukin 18, IL-18

350

S. Chen, et al. Schwann-like cells from ADSCs using folic acidFig 2. Morphological changes and expression of SC markers in SLCs (FA). (A) Microphotographs of H&E staining of ADSCs, SLCs (FA)and primary SCs (scale bar, 100 μm). (B) Relative mRNA expression of S100B (left) and p75NTR (right) in ADSCs, SLCs (FA) and primarySCs. (C) Confocal images showing the immunofluorescence staining for S100B and p75NTR in ADSCs, SLCs (FA) and primary SCs (scalebar, 200 μm). *P < 0.05 ; **P < 0.01, Mann-Whitney U test. adipose-derived stem cells, ADSCs ; Schwann cells, SCs ; Schwann-like cells,
SLCs ; folic acid, FA ; S100 calcium-binding protein B, S100B ; p75 neurotrophin receptor, p75NTR.

The Journal of Medical Investigation Vol. 68 August 2021

351

Fig 3. Relative expression of ErbB2 and EGR2 in different groups of SLCs. Relative mRNA expression of ERBB2 (A) and EGR2

(B) in conventional SLCs, SLCs (FA, 10 μg / ml), SLCs (FA) and SLCs (FA, 200 μg / ml) groups. **P < 0.01, Mann-Whitney U
test. adipose-derived stem cells, ADSCs ; Schwann-like cells, SLCs ; Erb-B2 receptor tyrosine kinase 2, ERBB2 ; early growth
response 2, EGR2 ; folic acid, FA.

Fig 4. Gene expression and secretion of NGF by ADSCs, SLCs and SCs. (A) Gene expression of NGF in ADSCs, different

groups of SLCs and SCs. (B) NGF concentration in the supernatants of ADSCs, different groups of SLCs, SCs and SCs (50μg / ml) groups. **P < 0.01, one way-ANOVA. adipose-derived stem cells, ADSCs ; Schwann-like cells, SLCs ; SCs ; Schwann-likecells ; folic acid, FA ; nerve growth factor, NGF.

DISCUSSION
Many studies have reported using differentiated SLCs fromvarious sources of stem cells to promote nerve regeneration(29-31). We focused on ADSCs, which exhibit several advantages : 1) a sufficient number of ADSCs can be easily obtained fromthe patient’s own adipose tissue through a minimally invasive
procedure ; 2) autologous ADSC transplantation poses fewer
ethical issues and lower risk of tumorigenicity compared withthe use of induced pluripotent or embryonic stem cells (32) ; and3) previous studies have shown excellent differentiation capabilities of ADSCs, including differentiation into powerful IPCs andhepatocyte-like cells (8, 33). However, the protocols used in thesestudies are similar, and an improved protocol to obtain more
useful SLCs requires further exploration.
This study used FA to induce stem cell differentiation. FA isa common water-soluble vitamin derivative. FA plays a key rolein the growth, differentiation and regeneration of the CNS (34)
and aids peripheral nerve repair via its neurotrophic effects.
Yilmaz et al. have shown that FA protects diabetic rats from diabetic peripheral neuropathy by reducing malondialdehyde levels
and upregulating NGF expression (35). Harma et al. have foundthat FA improved peripheral nerve healing and increased axon
myelination in a rat sciatic nerve injury model (17). Bridging the352

S. Chen, et al. Schwann-like cells from ADSCs using folic acidsciatic nerve defect using an FA-coated nerve conduit improved
the sciatic nerve function index and the nerve conduction velocity. Moreover, FA promoted proliferation, migration and NGF
secretion by SCs (18). Our results showed that the new differentiation cocktail induced a change in ASCD morphology into
bipolar and tripolar spindle shapes along with the expression of
typical SC markers, such as S100B and p75NTR, indicating effective differentiation into an SC phenotype.
After nerve injury, SCs transdifferentiate into so-called repair
SCs, and promote lysis of myelin, attract macrophages to participate in phagocytosis and secrete neurotrophic factors, such as
NGF, to support and control neuronal function and guide axonal
regeneration and axonal re-myelination (6, 28, 36, 37). Repair
SCs are considerably different from immature SCs. Immature
SCs fail to express mature SC markers, such as S100B andp75NTR. Studies have shown that ERBB2 may act as a maturation signal to negatively regulate the growth of axons, allowing
SCs to start re-myelination (38, 39). EGR2 is downregulated inrepair SCs and upregulated during re-myelination (28, 40). Interestingly, different conditions used to induce SLC differentiation determined the cell characteristics ; our new protocol seems
to generate repair SLCs. Our results show that the dose of FA
is related to the phenotypic characteristics of SLCs. Comparedwith 10 μg / ml and 200 μg / ml, the concentration of 50 μg / ml
could decrease the gene expression of both ERBB2 and EGR2.
There is little discussion in the literature on the state of the
SLCs ; it is reasonable to infer that the repair phenotype of SLCs
is the most beneficial for nerve regeneration when used to treat
PNI. NGF may activate autophagy in SCs, accelerate removal
and phagocytosis of myelin fragments, and promote regeneration of axons and myelin during the early stages of PNI (19).
Some studies have added neurotrophic factors, such as NGF,
into nerve conduits to improve the quality of nerve regeneration(41). Other studies have also changed the microenvironmentof transplanted cells, e.g., using self-assembling hydrogels, tostimulate higher secretion of brain-derived neurotrophic factors
and NGF (42). However, these approaches were limited by theshort half-life and unstable properties of neurotrophic factors
and by difficulties in preparing the graft. Therefore, there is anurgent need to find economical neurotrophic factors with long
half-lives and gradual release. According to our results, adding
FA into the differentiation protocol could promote the release of
NGF by SLCs. However, the secretion of NGF by SLCs (FA, 200
μg / ml) is not higher than SLCs (FA). We believe that due to thevulnerability of stem cells, excessive amount of FA might cause
potential damage to SLCs. So, we set the concentration of FA as
50 μg / ml in our new protocol.
However, this study still has some shortcomings as theses
SLCs (FA) generated by our modified protocol are still in conventional two-dimensional pattern. Many researchers including
our team (8, 9, 11, 43) have illustrated that the three-dimensional (3D) cell culture pattern could promote the differentiation
efficiency of stem cells and improve the final therapeutic effectof cell transplantation and cell therapy, which is considered tobe the trend of tissue engineering and regeneration medicine.
Therefore, we decide and have already begun to apply this new
protocol combined with 3D culture technology for the generationof more powerful SLCs and explore its potential on the nerve
regeneration in vivo and in vitro.

CONCLUSION
Our modified protocol effectively induced an SC phenotype in
ADSCs both in morphology and expression of SC markers. Furthermore, the addition of FA induced a repair phenotype typicalof SLCs with decreased ERBB2 and EGR2 gene expression andenhanced NGF secretion. This new protocol improved the functions of SLCs by using a stable and inexpensive vitamin and ispromising for SC-mediated nerve regeneration.

CONFLICTING INTERESTS
The authors declare no conflicts of interest.

ACKNOWLEDGEMENTS
This study was partly supported by a Grant-in-Aid for Scientific Research (grant no. 19K09045 to T. I., S. I., Y. M. and Y. S.,
and grant no. 19K09072 to T.T. and Y.S.). S. C., O. S., K. M., K.
T., S. Y., and M. S. declare no potential conflict of interest. We
thank Edanz Group (https://en-author-services.edanz.com/ac)
for editing a draft of this manuscript.

REFERENCES
1.

Hoke A : Mechanisms of disease : what factors limit the success of peripheral nerve regeneration in humans? Nat Clin
Pract Neurol 2 : 448-454, 2006
2. Pfister BJ, Gordon T, Loverde JR, Kochar AS, Mackinnon
SE, Cullen DK : Biomedical engineering strategies for peripheral nerve repair : surgical applications, state of the art,and future challenges. Crit Rev Biomed Eng 39 : 81-124,
2011
3. Wallis CJD, Peltz S, Byrne J, Kroft J, Karanicolas P,
Coburn N, Nathens AB, Nam RK, Hallet J, Satkunasivam
R : Peripheral Nerve Injury during Abdominal-Pelvic Surgery : Analysis of the National Surgical Quality Improvement Program Database. Am Surg 83 : 1214-1219, 2017
4. Xia WZ, Zhu J, Wang XY, Tang YD, Zhou P, Hou M,
Li ST : ANXA1 directs Schwann cells proliferation andmigration to accelerate nerve regeneration through the
FPR2 / AMPK pathway. FASEB J 34 : 13993-14005, 2020
5. Xia WZ, Zhu J, Wang XY, Tang YD, Zhou P, Wei XY, Chang
BW, Zheng X, Zhu WC, Hou M, Li ST : Overexpression of
Foxc1 regenerates crushed rat facial nerves by promoting
Schwann cells migration via the Wnt / β-catenin signaling
pathway. J Cell Physiol 235 : 9609-9622, 2020
6. Rhode SC, Beier JP, Ruhl T : Adipose tissue stem cells in peripheral nerve regeneration-In vitro and in vivo. J Neurosci
Res 99 : 545-560, 2021
7. Ikemoto T, Feng R, Shimada M, Saito Y, Iwahashi S, Morine
Y, Imura S : A New 2-Step Acceleration Protocol Using aHistone Deacetylase Inhibitor to Generate Insulin-Producing Cells From Adipose-Derived Mesenchymal Stem Cells.
Pancreas 47 : 477-481, 2018
8. Ikemoto T, Feng R, Iwahashi SI, Yamada S, Saito Y, Morine
Y, Imura S, Matsuhisa M, Shimada M : In vitro and in vivo
effects of insulin-producing cells generated by xeno-antigen
free 3D culture with RCP piece. Sci Rep 9 : 10759, 2019
9. Wada Y, Ikemoto T, Morine Y, Imura S, Saito Y, Yamada S,
Shimada M : The Differences in the Characteristics of Insulin-producing Cells Using Human Adipose-tissue Derived
Mesenchymal Stem Cells from Subcutaneous and VisceralTissues. Sci Rep 9 : 13204, 2019
10. Ohta S, Ikemoto T, Wada Y, Saito Y, Yamada S, Imura S,
Morine Y, Shimada M : A change in the zinc ion concentration reflects the maturation of insulin-producing cells generated from adipose-derived mesenchymal stem cells. Sci Rep

The Journal of Medical Investigation Vol. 68 August 2021
9 : 18731, 2019
11. Tokuda K, Ikemoto T, Saito Y, Miyazaki K, Yamashita S,
Yamada S, Imura S, Morine Y and Shimada M : The Fragility of Cryopreserved Insulin-producing Cells Differentiatedfrom Adipose-tissue-derived Stem Cells. Cell Transplant
29 : 963689720954798, 2020
12. Kingham PJ, Kalbermatten DF, Mahay D, Armstrong SJ,
Wiberg M, Terenghi G : Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite
outgrowth in vitro. Exp Neurol 207 : 267-274, 2007
13. Kim GB, Chen YJ, Kang WB, Guo JS, Payne R, Li H, Wei
Q, Baker J, Dong C, Zhang SL, Wong PK, Rizk EB, Yan JZYang J : The critical chemical and mechanical regulation offolic acid on neural engineering. Biomaterials 178 : 504-516,
2018
14. Iskandar BJ, Nelson A, Resnick D, Skene JHP, Gao P,
Johnson C, Cook TD, Hariharan N : Folic acid supplementation enhances repair of the adult central nervous system.
Ann Neurol 56 : 221-227, 2004
15. Davis CK, Rajanikant G K : Postischemic supplementationof folic acid improves neuronal survival and regeneration invitro. Nutr Res 75 : 1-14, 2020
16. Weng QJ, Wang JJ, Wang JY, Tan BQ, Wang J, Wang
HB, Zheng T, Lu QR, Yang B, He QJ : Folate Metabolism
Regulates Oligodendrocyte Survival and Differentiation by
Modulating AMPKα Activity. Sci Rep 7 : 1705, 201717. Harma A, Sahin MS, Zorludemir S : Effects of intraperitoneally administered folic acid on the healing of repaired tibial nerves in rats. J Reconstr Microsurg 31 : 191-197, 2015
18. Kang WB, Chen YJ, Lu DY, Yan JZ : Folic acid contributes
to peripheral nerve injury repair by promoting Schwann
cell proliferation, migration, and secretion of nerve growth
factor. Neural Regen Res 14 : 132-139, 201919. Li R, Li DH, Wu CB, Ye LB, Wu YQ, Yuan Y, Yang SN, Xie
L, Mao YQ, Jiang T, Li YY, Wang J, Zhang HY, Li XK, Xiao
J : Nerve growth factor activates autophagy in Schwann
cells to enhance myelin debris clearance and to expedite
nerve regeneration. Theranostics 10 : 1649-1677, 2020
20. Tao HY, He B, Liu SQ, Wei AL, Tao FH, Tao HL, Deng WX,
Li HH, Chen Q : Effect of carboxymethylated chitosan on
the biosynthesis of NGF and activation of the Wnt / β-cateninsignaling pathway in the proliferation of Schwann cells. Eur
J Pharmacol 702 : 85-92, 2013
21. Gu XK, Li XR, Lu ML, Xu H : Lithium promotes proliferation and suppresses migration of Schwann cells. Neural
Regen Res 15 : 1955-1961, 2020
22. Wang HK, Zhang P, Yu J, Zhang FC, Dai WZ, Yi S : Matrix metalloproteinase 7 promoted Schwann cell migrationand myelination after rat sciatic nerve injury. Mol Brain
12 : 101, 201923. Chang HM, Liu CH, Hsu WM, Chen LY, Wang HP, Wu TH,
Chen KY, Ho WH, Liao WC : Proliferative effects of melatonin on Schwann cells : implication for nerve regeneration
following peripheral nerve injury. J Pineal Res 56 : 322-332,
2014
24. Piovesana R, Faroni A, Taggi M, Matera A, Soligo M,
Canipari R, Manni L, Reid AJ, Tata AM : Muscarinic receptors modulate Nerve Growth Factor production in rat
Schwann-like adipose-derived stem cells and in Schwann
cells. Sci Rep 10 : 7159, 2020
25. Piovesana R, Faroni A, Magnaghi V, Reid AJ, Tata AM : M2
receptors activation modulates cell growth, migration anddifferentiation of rat Schwann-like adipose-derived stem
cells. Cell Death Discov 5 : 92, 201926. Tzekova N, Heinen A, Bunk S, Hermann C, Hartung H-P,
Reipert B, Küry P : Immunoglobulins stimulate cultured

353

Schwann cell maturation and promote their potential to induce axonal outgrowth. J Neuroinflammation 12 : 107, 2015
27. Garratt AN, Voiculescu O, Topilko P, Charnay P, Birchmeier
C : A dual role of erbB2 in myelination and in expansion of
the schwann cell precursor pool. J Cell Biol 148 : 1035-1046,
2000
28. Nocera G, Jacob C : Mechanisms of Schwann cell plasticity
involved in peripheral nerve repair after injury. Cell Mol
Life Sci 77 : 3977-3989, 2020
29. Bayat N, Ebrahimi-Barough S, Ardakan MMM, Ai A,
Kamyab A, Babaloo H, Ai J : Differentiation of Human
Endometrial Stem Cells into Schwann Cells in Fibrin Hydrogel as 3D Culture. Mol Neurobiol 53 : 7170-7176, 2016
30. Jung N, Park S, Choi Y, Park J-W, Hong YB, Park HHC,
Yu Y, Kwak G, Kim HS, Ryu K-H, Kim JK, Jo I, Choi
B-O, Jung S-C : Tonsil-Derived Mesenchymal Stem Cells
Differentiate into a Schwann Cell Phenotype and Promote
Peripheral Nerve Regeneration. Int J Mol Sci 17 : 1867, 2016
31. Liu YT, Chen JH, Liu W, Lu XC, Liu ZY, Zhao XB, Li GC,
Chen ZB : A Modified Approach to Inducing Bone Marrow Stromal Cells to Differentiate into Cells with Mature
Schwann Cell Phenotypes. Stem Cells Dev 25 : 347-359,
2016
32. Mizuno H : Adipose-derived stem and stromal cells for cellbased therapy : current status of preclinical studies andclinical trials. Curr Opin Mol Ther 12 : 442-449, 2010
33. Saito Y, Ikemoto T, Morine Y, Shimada M : Current statusof hepatocyte-like cell therapy from stem cells. Surg Today
51 : 340-349, 2021.
34. Balashova OA, Visina O, Borodinsky LN : Folate action in
nervous system development and disease. Dev Neurobiol
78 : 391-402, 201835. Yilmaz M, Aktug H, Oltulu F, Erbas O : Neuroprotective
effects of folic acid on experimental diabetic peripheral neuropathy. Toxicol Ind Health 32 : 832-840.
36. Frostick SP, Yin Q, Kemp GJ : Schwann cells, neurotrophic
factors, and peripheral nerve regeneration. Microsurgery
18 : 397-405, 1998
37. Bunge RP : Expanding roles for the Schwann cell : ensheathment, myelination, trophism and regeneration. Curr
Opin Neurobiol 3 : 805-809, 1993
38. Hendry JM, Alvarez-Veronesi MC, Placheta E, Zhang JJ,
Gordon T, Borschel GH : ErbB2 blockade with Herceptin(trastuzumab) enhances peripheral nerve regeneration
after repair of acute or chronic peripheral nerve injury. Ann
Neurol 80 : 112-126, 2016
39. Citri A, Skaria KB, Yarden Y : The deaf and the dumb : thebiology of ErbB-2 and ErbB-3. Exp Cell Res 284 : 54-65,
2003
40. Liu ZY, Jin YQ, Chen LL, Wang Y, Yang XN, Cheng J, Wu
W, Qi ZL, Shen ZL : Specific marker expression and cellstate of Schwann cells during culture in vitro. PLoS One
10 : e0123278, 2015
41. Takagi T, Kimura Y, Shibata S, Saito H, Ishii K, Okano HJ,
Toyama Y, Okano H, Tabata Y, Nakamura M : SustainedbFGF-release tubes for peripheral nerve regeneration : comparison with autograft. Plast Reconstr Surg 130 : 866-876,
2012
42. Faroni A, Workman VL, Saiani A, Reid AJ : Self-Assembling Peptide Hydrogel Matrices Improve the Neurotrophic
Potential of Human Adipose-Derived Stem Cells. Adv
Healthc Mater 8 : e1900410, 201943. Yu CP, Juang JH, Lin YJ, Kuo CW, Hsieh LH, Huang
CC : Enhancement of subcutaneously transplanted β cell
survival using 3D stem cell spheroids with proangiogenic
and prosurvival potential. Adv. Biosys 4 : e1900254, 2020