Invest Clin 64(3): 267 - 280, 2023 https://doi.org/10.54817/IC.v64n3a1

Corresponding author: Li Lu. School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, China;

Medical College of Lanzhou University, Cheng guan District, Lanzhou city. Gansu, China. Telephone: 86+ 0931-

8915023. E-mail: lul@lzu.edu.cn

Induced differentiation of adipose-derived

stem cells enhance secretion of neurotrophic

factors.

Xin Zeng

, Ya-nan Liu

, Zhen Li

, Yun He

, Fang Li

, Shu-yuan Zhang

, Jing Gu

and Li Lu

1,3

School of Basic Medical Sciences, Lanzhou University, Lanzhou, Gansu, China.

The First Clinical Medical College, Lanzhou University, Lanzhou, Gansu, China.

Gansu University of Traditional Chinese Medicine, Lanzhou, Gansu, China.

Keywords: differentiated ADSCs; Schwann cells; neurotrophic factors; P2X7; nerve

damage.

Abstract. Adipose-derived stem cells (ADSCs) could be ideal seed cells

for repairing nerve injury as they have the potential for multidirectional dif-

ferentiation. However, it is still unclear whether the undifferentiated or the

differentiated ADSCs have priorities in promoting axonal regeneration and my-

elin formation. In this study, the primary ADSCs from rats were cultured and

differentiated. The morphology, differentiation potential, and secretion of neu-

rotrophic factors of ADSCs were compared before and after induction. Undiffer-

entiated ADSCs (uADSCs) were aggregated into bundles containing reticular,

star, and polygonal structures. They contained a large number of lipid droplets

and were positive for Oil red O staining. After differentiation, differentiation

ADSCs (dADSCs) become long and spindle-shaped with decreasing protrusions

around the cells, spiraling growth, and were negative for Oil red O staining.

When comparing the groups the flow cytometer analysis showed: similar CD29

and CD45 surface markers in both groups; and CD44 and CD90 markers were

very low in the undifferentiated groups. The levels of neurotrophin 3 (NT-3)

and neuregulin 1 (NRG-1), and their receptors tropomyosin receptor kinase C

(TrkC) and receptor protein-tyrosine kinase erbB-4 (ErbB-4) in dADSCs were

higher than those in uADSCs. While the expressions of myelin protein zero

(P0), myelin-associated glycoprotein (MAG), and purine receptor P2X7 (P2X7)

were not significantly different before and after differentiation. It may be specu-

lated that the dADSCs have enhanced abilities in nerve repairment which is

associated with increased expression of neurotrophic factors.

268 Zeng et al.

Investigación Clínica 64(3): 2023

La diferenciación inducida de las células madre derivadas del

tejido adiposo aumenta la secreción de factores neurotróficos.

Invest Clin 2023; 64 (3): 267 – 280

Palabras clave: ADSC diferenciadas, células de Schwann, factores neurotróficos, P2X7,

daño nervioso.

Resumen. Las células madre derivadas del tejido adiposo (ADSCs) podrían

ser una semilla ideal de células para la reparación de lesiones nerviosas, ya

que tienen el potencial de diferenciación multidireccional. Sin embargo, aún

no está claro si las ADSCs indiferenciadas o diferenciadas tienen prioridades

en la promoción de la regeneración axonal y la formación de mielina. En este

estudio, ADSCs primarias de las ratas fueron cultivadas y diferenciadas. Se com-

pararon la morfología, el potencial de diferenciación y la secreción de los fac-

tores neurotróficos de las ADSCs antes y después de la inducción. Las ADSCs

indiferenciadas (uADSCs) se encontraban agregadas en haces que contenían

estructuras reticulares, estrelladas y poligonales. Contenían un gran número

de gotitas de lípidos y fueron positivas para la tinción de Aceite Rojo O. Después

de la diferenciación, las ADSCs (dADSCs) se vuelven largas y en forma de huso

con un número decreciente de protuberancias alrededor de las células, creci-

miento en espiral, y fueron negativas para la tinción de Aceite Rojo O. Cuando

se compararon los dos grupos, análisis del citómetro de flojo muestra que los

dos grupos de marcadores superficiales CD29 y CD45 eran similares; y los mar-

cadores CD44 y CD90 eran muy bajos en el grupo indiferenciado. Los niveles de

neurotrofina 3 (NT-3) y neuregulina 1 (NRG-1) y sus receptores, el receptor de

tropomiosina quinasa C (TrkC) y el receptor de proteína tirosina quinasa erbB-

4 (ErbB-4) en dADSC fueron más altos que los de uADSC. Mientras que las

expresiones de proteína cero de mielina (P0), glicoproteína asociada a mielina

(MAG) y receptor de purina P2X7 (P2X7) no fueron significativamente dife-

rentes antes y después de la diferenciación. Se puede especular que las dADSC

tienen capacidades mejoradas en la reparación nerviosa que se asocia con una

mayor expresión de factores neurotróficos.

Received: 10-11-2022 Accepted: 03-04-2023

INTRODUCTION

Regeneration and functional recovery

after peripheral nerve damage are foci of

research in neuroscience and a problem in

clinical surgery

. Schwann cells (SCs) are

the primary myelin-forming cells in the pe-

ripheral nervous system and play a promi-

nent role in neuron survival and function.

Schwann cells promote nerve regeneration

by secreting neurotrophic factors and adhe-

sion molecules

. However, the clinical use of

Schwann cells is limited by the difficulty in

obtaining adequate quantities. To overcome

this, the ability of various types of stem cells

to differentiate into Schwann cells is under

investigation. Stem cells as seed cells com-

bined with vector scaffolds can be used to

Differentiation of adipose-derived stem cells enhance secretion of neurotrophic factors 269

Vol. 64(3): 267 - 280, 2023

construct tissue-engineered nerves with bi-

ological activity and functionality; indeed,

this research focuses on peripheral nerve

repair. Adipose-derived stem cells (ADSCs)

can be induced to differentiate into nerve

cells

, astrocytes, osteoblasts, and myofibro-

blasts in the appropriate type of medium

4-9

Adipose tissue has the largest storage capac-

ity in the body and is easily harvested and

cultured. ADSCs are genetically stable, have

low tumorigenicity, low immunogenicity,

and show rapid expansion in vitro

. There-

fore, ADSCs can be used to repair peripheral

nerve damage.

ADSCs can be induced to differentiate

into Schwann-like cells in vitro, which in-

volves a changing from a flat to an elongated

spindle shape and expressing s-100, GFAP,

and P75. In coculture with spinal dorsal root

ganglion (DRG) neurons, induced ADSCs

promoted the axonal growth of DRG neurons

and myelin sheath formation

11,12

, indicating

that the induced ADSCs had the phenotype

and functionality of Schwann cells.

Repair by ADSCs of injured nerves has

been confirmed in vivo. In mice with sciatic

nerve injury, intravenous injection of ADSCs

significantly increased the growth of sciatic

nerve axons and ameliorated the inflamma-

tory response

. ADSCs were transferred

into artificial nerve conduits made of colla-

gen

, silica gel

8,9

, PCL

, and fibroin/col-

lagen

and transplanted into sciatic nerve

defects of rats. The regenerated axon of the

sciatic nerve in the transplantation group

was longer, and the walking gait, muscle

weight, and nerve conduction velocity were

significantly improved compared to that in

the control group

7,15-17

The mechanism by which ADSCs repair

peripheral nerve injury is unclear. After dif-

ferentiation, the mRNA and protein levels of

the purine receptor P2X7 increased signifi-

cantly in ADSCs, stimulating Ca2+ inflow

and inhibiting the P2X7 receptor to prevent

ATP-induced cell death

The therapeutic effect and the under-

lying mechanisms of uADSCs and dADSCs

on nerve injury are unclear. It is crucial to

determine the phenotypic changes of AD-

SCs before and after differentiation is in-

duced. Repair of peripheral injured nerves

involves the secretion of neurotrophic fac-

tors, axon growth, and myelin sheath forma-

tion. Schwann cells secrete multiple growth

factors that promote axonal regeneration,

including nerve growth factor (NGF), brain-

derived neurotrophic factor (BDNF), and

neurotrophin-3 (NT-3)

. Stem cells repair

damaged tissue by releasing several trophic

factors in situ, which alters the local micro-

environment

. To compare the efficacy of

uADSCs and dADSCs in treating peripheral

nerve injury, we investigated the effect of

induction of differentiation on the differen-

tiation potential, morphology, proliferation,

and levels of Schwann cell-related proteins

of ADSCs.

MATERIALS AND METHODS

Extraction, isolation, and culture

of ADSCs

Male Sprague-Dawley (SD) specific-

pathogen-free rats, of approximately 300 g of

weight were euthanized by cervical disloca-

tion and soaked in 75% ethanol for 10 min.

Adipose tissue under the skin of the abdo-

men was dissected and placed in a Petri dish

containing phosphate-buffered saline (PBS,

pH=7.2) (Solarbio, Beijing, China). The cells

were washed with PBS three times to remove

blood and vessels. The adipose tissue was cut

into one mm

pieces, digested with 0.075%

type I collagenase (Invitrogen, Carlsbad, Cali-

fornia, USA), and incubated at 37°C for 90min.

After centrifugation, the upper undigested

adipose tissue and the supernatant were dis-

carded, and the pellet was washed in Dulbec-

co’s modified Eagle’s medium (DMEM)/F12

(containing 1% penicillin/streptomycin and

10% fetal bovine serum). The cells were cen-

trifuged, filtered through a 200-mesh sieve,

and transferred to a 75cm

culture flask. Af-

ter 24h, half of the medium was replaced, and

the cells were passaged at a ratio of 1:2. Then

270 Zeng et al.

Investigación Clínica 64(3): 2023

they were cultured to the third generation to

achieve the purity of the isolated cells, and the

cell morphology (CKX41, Olympus, Tokyo,

Japan) was examined. When the cells were

grown at their best, they were digested and

centrifuged with 0.25% trypsin, and collected

in DMSO: FBS: DMEM/F12=1:2:7, and mixed

with cryopreserved solution, which was added

into the cryopreservation tube after resus-

pension, and placed at 4°C for 1 hour, -20°C

for 4 hours, and -80°C overnight. Finally, they

were transferred to the liquid nitrogen tank

for cryopreservation for later use. The experi-

ment was conducted in three batches of cells.

Cell viability assay

Cell viability was evaluated by the

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

tetrazolium bromide (MTT) assay. ADSCs

were cultured in a 96-well plate, and 20µL

(0.6mM) of MTT solution was added to each

well. The cells were incubated at 37°C for

4h, 150µL of dimethyl sulfoxide was added

to each well, and the plates were shaken

for 10 min to ensure that crystals were dis-

solved. The optical density was measured at

570nm. The experiment was conducted in

three batches of cells.

Induction of differentiation of ADSCs

We hypothesized that adipose-derived

stem cells are more conducive to axonal

regeneration and myelination after differ-

entiation. In order to verify this, this study

mainly investigated the differences between

undifferentiated and differentiated ADSCs

in cell morphology and in promoting neuro-

trophic factor secretion and myelin-related

protein expression. ADSCs were digested

with 0.25% trypsin/ EDTA (Invitrogen, USA)

at the third passage, centrifuged, resus-

pended in DMEM/F12, and transferred to a

six-well plate (2×10

/mL). After 24h, 2mL

of 1mM β-mercaptoethanol (Sigma-Aldrich,

USA) was added, followed by fresh medium

containing 35ng/mL all-trans-retinoic acid

(Sigma-Aldrich, USA). The cells were washed

in PBS after cultivation for 72h, and 2mL

of ADSC differentiation medium (DMEM/

F12 containing 5 ng/mL platelet-derived

growth factor, 10 ng/mL basic fibroblast

growth factor, 14µM forskolin, and 200ng/

mL heregulin) was added. The experiment

was conducted in three batches of cells, the

cells were maintained for 1 week under the

same conditions and fresh medium was add-

ed at 48–72h intervals. The morphological

characteristics of uADSCs and dADSCs were

observed under a microscope.

Oil red O staining

uADSCs and dADSCs were cultured in

six-well plates (2×10

/mL), the medium was

discarded, and the cells were washed in PBS

three times and fixed in 10% formaldehyde

for 40min. Next, the cells were washed three

times in PBS, stained with Oil Red O (World-

bio, China), and incubated at room tempera-

ture for 40min. The cells were rinsed with

75% alcohol to remove excess dye. The cells

were sealed with glycerin gelatin, and cell

morphology was observed under a micro-

scope. Three batches of cell morphological

maps were collected and analyzed.

Flow cytometry

uADSCs and dADSCs were digested

with 0.25% trypsin/EDTA, centrifuged, and

the supernatant was discarded. The cells

were washed three times in 2% bovine serum

albumin (BSA; abcbio, China). The pellet

was resuspended in 5% BSA and subjected to

cell counting. The cell density was adjusted

to 1×10

/mL, and the cells were incubat-

ed with 10µL of antibodies against CD29,

CD44, CD90, and CD45 (Bio-Rad) for 30min

on ice in the dark. The cells were washed in

5% BSA, centrifuged (1500 rpm) for 5min,

and resuspended in PBS for flow cytometry.

Three batches of cells were collected and

analyzed.

Western blotting

Cells were rinsed in 0.01M PBS and

lysed in radioimmunoprecipitation assay ly-

sis buffer. The lysates were centrifuged at

Differentiation of adipose-derived stem cells enhance secretion of neurotrophic factors 271

Vol. 64(3): 267 - 280, 2023

4°C at 12,000 rpm for 5min. The superna-

tant was collected and stored at -20°C. The

protein concentration was quantified using

a BCA Protein Assay Kit (Beyotime Biotech-

nology, China). The proteins were resolved

by sodium dodecyl sulfate-polyvinylidene

fluoride gel electrophoresis and transferred

onto polyvinylidene fluoride membranes. Pri-

mary antibodies against NRG-1 (1:1000, Af-

finity), NT-3 (1:500, Servicebio), P0 (1:500,

Affinity), MAG(1:1000,Bioss), TrkC (1:500,

GeneTex), ErbB-4 (1:500, GeneTex), and

P2X7 (1:1000, ab109054, Abcam), were

added and the membranes were incubated

at 4°C for 24h. Next, the secondary anti-

body was added, followed by incubation for

2h.β-actin was used as the loading control

for normalization. The grey values of the tar-

get protein were analyzed by Image J. The

results were calculated by the ratio of accu-

mulated gray values of target protein to the

bands of β-actin, which represented the rela-

tive expression level of target protein. Bands

of each target protein where appeared three

times, were sorted out and analyzed.

Statistical analysis

Prism 5.0 was used for statistical analysis

(GraphPad Systems, Inc., La Jolla, CA, USA),

performed by t test. The data are means ±

standard deviation. P<0.05 was considered

indicative of statistical significance.

RESULTS

Morphological characteristics of ADSCs

ADSCs were isolated from subcutaneous

abdominal fat of male SD rats and cultured

in DMEM/F12. After 48h, the cells began to

grow rapidly (Fig. 1A), adhered to the wall,

and formed a short fusiform, star-shaped

structure and irregular polygonal structure.

After 5–7 days, the ADSCs were spindle-

shaped and growing vigorously (Fig. 1B).

Growth of ADSCs

The ADSC growth curve was S-shaped.

The ADSCs entered the logarithmic growth

phase after 72h, and growth peaked at day

5 and decreased thereafter (Fig. 1C). In ad-

dition, ADSCs at passage 3 had the highest

growth rate and those at passage 13 the low-

Fig. 1. Morphological characteristics and proliferation changes of ADSCs. A. After 48h, ADSCs were short

and fusiform, with a star-shaped structure and irregular polygonal structure (×10). B. After 5–7 days,

the primary ADSC cells exhibited a typical fibroblast-like morphology (×10). C. Growth curves of

ADSCs at passages 3, 5, 7, 9 and 13. D. Growth curves before and after cryopreservation (cADSCs,

ADSCs after cryopreservation).

272 Zeng et al.

Investigación Clínica 64(3): 2023

est; therefore, the growth rate decreased

with increasing passage number. At the

initial stage of culture, the growth rate of

cryogenically preserved ADSCs was similar

to that of freshly prepared ADSCs (Fig. 1D).

Morphological characteristics of uADSCs

induced to differentiate into dADSCs

The uADSCs aggregated into bundles

containing reticular, star structures, and po-

lygonal structures (Fig. 2A). After differenti-

ation the cells were long and spindle-shaped,

the number of protrusions around the cells

decreased, spiraling growth, and showing a

Schwann-like morphology (Fig. 2B).

Oil red O staining

The uADSCs contained a small number

of lipid droplets (Fig. 3A). The dADSCs are

Schwann cell-like cells and did not exhibit

lipid droplets (Fig. 3B).

Expression of cell surface factors

Flow cytometry was performed to exam-

ine CD29, CD44, CD90 (stem-cell markers),

and CD45 expression levels. In ADSCs, 96.0%

expressed CD29, 60.1% expressed CD44, and

74.2% expressed CD90, indicating ADSCs have

mesenchymal stem cell-related surface mark-

ers and have the potential of multi-differen-

tiation of stem cells (Fig.4). As a marker of

hematopoietic cells, the positive rate of CD45

was less than 50% (only 32.5%), suggesting

they were uADSCs but could not differentiate

into hematopoietic cells. After induction and

differentiation into SCs, in dADSCs, 99.1% ex-

pressed CD29, 91.4% expressed CD44, 96.8%

expressed CD90, and only 31.2% expressed

CD45 (Fig.4). Compared with uADSCs, the ex-

pression levels of each marker in dADSCs were

significantly increased, suggesting that the dif-

ferentiation potential of ADSCs induce to dif-

ferentiate into SCs was enhanced (Fig. 5).

Fig. 2. Morphological changes of ADSCs after induced differentiation. A. Morphology of uADSCs. B. Morpho-

logy of dADSCs. After induction, the cells were long and spindle-shaped, the number of protrusions

around the cells decreased, and spiraling growth (×10).

Fig. 3. Morphological changes after Oil red O staining. A.Oil red O staining of uADSCs. B.Oil red O staining

of dADSCs (×10).

Differentiation of adipose-derived stem cells enhance secretion of neurotrophic factors 273

Vol. 64(3): 267 - 280, 2023

Fig. 4. CD29, CD44, CD45, and CD90 expression in uADSCs and dADSCs by flow cytometry. A. Expression

of CD29, CD44, CD90, and CD45 in uADSCs. B. Expression of CD29, CD44, CD90, and CD45 in

dADSCs. Blank control, cells treated with PBS but not anti-CD antibody.

274 Zeng et al.

Investigación Clínica 64(3): 2023

Expression of uADSC- and dADSC-related

proteins

Western blotting showed that NRG-1,

ErbB-4, NT3, TrkC, P0, MAG and P2X7 were

expressed on the surface of uADSCs and

dADSCs (Fig. 6). The levels of NRG-1, ErbB4,

NT-3, and TrkC in dADSCs were significant-

ly higher than those in uADSCs (p<0.05).

However, there were no significant differ-

ences in the levels of the myelin protein P0,

MAG, and the purine receptor P2X7 before

and after induction of differentiation.

DISCUSSION

Mesenchymal stem cells (also known as

all-powerful mesenchymal stromal cells), as

one of the stem cells, have attracted much

attention in the field of stem cell therapy

and regenerative medicine. ADSCs play an

Fig. 5. Expression levels of CD29, CD44, CD45, and CD90 in uADSCs were lower than those in dADSCs (*p

<0.05, n = 3).

Fig. 6. Changes in NRG-1, NT3, TrkC, ErbB-4, P0, MAG and P2X7 protein levels. β-actin was used as the loa-

ding control (*p<0.05, n = 3).

Differentiation of adipose-derived stem cells enhance secretion of neurotrophic factors 275

Vol. 64(3): 267 - 280, 2023

important role in nerve injury and function-

al recovery and are considered ideal seed

cells for nerve transplantation. However, it

is currently unclear whether ADSCs differ-

entiate into dADSCs after being stimulated

by the in vivo injury environment or their

direct effects on promoting nerve regenera-

tion

. In this study, type I collagenase was

used to isolate the subcutaneous adipose tis-

sue of rat abdomen to evaluate the differen-

tiation potential, morphology, and protein

levels before and after differentiation. When

the cells passed to the third generation, the

cell growth activity was the best, so the third

generation of ADSCs was selected as the re-

search object. Cryopreservation is used for

long-term storage of biological materials,

such as oocytes, stem cells, vascular tissues,

and embryos

21-23

. In this study, revived AD-

SCs undergoing cryopreservation did not

show significant loss of viability or prolifera-

tion by MTT assay.

Some scholars compared the roles of

dADSCs and uADSCs in nerve transplanta-

tion and believed that undifferentiated adi-

pose stem cells (ADSCs) were easy to obtain

and had the advantage of a short culture

cycle in promoting neurotrophic factors se-

cretion and repairing myelin sheath injury

24,25

. At the same time, studies have pointed

out that differentiated adipose stem cells

are more conducive to playing the role of

stem cells in nerve injury repair

. In this

study, adipose stem cells were differentiated

and cultured to explore the advantages and

disadvantages of undifferentiated and differ-

entiated ADSCs in promoting axonal regen-

eration and myelination. The morphologies

of uADSCs and dADSCs were significantly

different. uADSCs cells presented star and

polygonal structures. After 1-2 w of induc-

tion and differentiation by adding specific

Schwann-inducing fluid, dADSCs cells pre-

sented a spindle shaped Schwann-like cell

morphology, and the number of protuber-

ances around the cells decreased. ADSCs ex-

pressed mesenchymal stem cell (MSC) mark-

ers and have similar properties to MSCs

MSCs derived from some mammals can

be transformed into Schwann cells in the

presence of inducers or mixtures of growth

factors. ADSCs express several stem cell sur-

face molecules such as CD105, CD29, CD44,

and CD45

. In the recently published stud-

ies, just like our method, only positive mark-

ers were detected in the differentiation and

identification of primary ADSCs cells, less in-

volving negative markers such as CD34

27, 28

High expression of CD29, CD44, and CD90

and low expression of CD45 were found in

uADSCs and dADSCs. Jiang et al.

reported

that ADSCs isolated from SD mice show high

CD29, CD44, and CD90 expression levels,

but low or absent expression of CD45. This

agrees with our finding that both uADSCs

and dADSCs express MSC-associated surface

markers and can undergo differentiation

into multiple cell lineages. The uADSCs, but

not the dADSCs, were positive for Oil red O

staining, indicating that the former had a

larger number of lipid droplets than the lat-

ter.

Repairing and regenerating damaged

nerves involves a complicated pathophysi-

ological process, mainly dependent on regu-

lating various cytokines. When injured, the

body can rely on its own nerve regeneration

or granulation tissue hyperplasia and scar

formation to achieve healing. ADSCs partici-

pate in various stages of tissue repair by vir-

tue of their multiple physiological functions

. Multidirectional studies have confirmed

that ADSCs can secrete various cytokines,

which play a vital role in diverse physiologi-

cal activities of ADSCs.

Schwann cells (SCs) are the most prom-

ising seed cells for peripheral nerve tissue

engineering

, which can promote peripher-

al axon regeneration after peripheral nerve

injury (PNI). Recent research has found that

salidroside may improve the regeneration

effect on the sciatic nerve following a com-

bined application of epimysium conduit and

RSC96 Schwann cells in rats

. Epothilone

B (EpoB) is an FDA-approved antineoplastic

agent, which shows the capacity to induce

276 Zeng et al.

Investigación Clínica 64(3): 2023

alpha-tubulin polymerization and improve

microtubules’ stability. The latest research

found the potential therapeutic value of

EpoB in enhancing regeneration and func-

tional recovery in cases of PNI

. In addition,

Schwann cells also play a significant role in

promoting the regeneration of PNI. Accord-

ing to the latest research, SCs are integral

in the regeneration and restoration of func-

tion following PNI. SCs are able to dediffer-

entiate and proliferate, remove myelin and

axonal debris, and are supportive of axonal

regeneration

. Moreover, 5% gastrodin/PU

NGC efficiently promotes nerve regenera-

tion, indicating their potential for use in pe-

ripheral nerve regeneration applications

Schwann cells (SCs) secrete neuro-

trophin 3 (NT-3). NT-3 can promote the de-

velopment and differentiation of neurons,

and its binding with tropomyosin receptor

kinase C (TrkC) receptor can maintain the

survival of neurons

, inhibit cell apoptosis,

and promote the differentiation of SCs into

neurons

36,37

. Several studies have shown that

in vitro transfection of adenovirus carrying

NT-3 (AdvNT-3) gene can promote the dif-

ferentiation of MSCs into neuron-like cells.

The role of NT-3 is mediated by its preferred

binding receptor TrkC. In this study, the

expression levels of NT-3 and TrkC were sig-

nificantly increased after the induction of

differentiation of ADSCs, consistent with

the above. Western blot results showed that

dADSCs could promote the expression of

NT-3 and its receptor TrkC, thus maintain-

ing the regeneration of injured nerves and

reducing nerve apoptosis.

The neuregulin-1 (NRG-1)/receptor/

tyrosine protein kinase ErbB (ErbB) system

is an endothelium-controlled paracrine sys-

tem. It has been found that NRG-1 can pro-

mote the recovery of nerve function after

brachial plexus injury after contralateral C7

nerve root metastasis in rats, and NRG-1 has

combined anti-inflammatory and anti-fibro-

sis effects in different organs, including skin,

lung, and heart. There is increasing evidence

that the NRG-1/ErbB system is active in var-

ious organs throughout the body. NRG-1 not

only promotes neuronal activity but also acts

on the receptor ErbB-4 in nerve endings. At

nerve endings, NRG-1 enters the cell body

through axoplasmic countercurrent and pro-

motes the growth of neurons. In this study,

NRG-1 and erbB-4 expression levels were sig-

nificantly increased after induction of ADSC

differentiation, consistent with a previous

report

. Thus, the ability of dADSCs to se-

crete neurotrophic factors and promote the

growth of axons likely explains their ability

to repair peripheral nerve injury.

Repair of injured nerves is accompanied

by myelin sheath formation, which involves

the coordinated synthesis of a group of pro-

teins related to myelin, including the trans-

membrane glycoprotein P0 and myelin-as-

sociated glycoprotein MAG. MAG is a major

component of myelin-derived nerve growth

inhibitor. MAG shows different functions at

different stages of the nervous system de-

velopment, promoting axon growth during

development and inhibiting axon growth

during maturation. In this study, the level

of the myelin-sheath protein P0 and MAG in

uADSCs and dADSCs was not significantly

different before and after induction of differ-

entiation. Synthesis by Schwann cells of P0

is dependent on contact with axons

. The

synthesis of P0 in Schwann cells is regulated

by neural developmental growth

. There-

fore, we can speculate that our results may

be related to this cause.

P2X7 is a non-selective cationic channel

receptor expressed in neurons and smooth

muscle; the ligand of this receptor is ATP

. P2X7 acts as a bridge between the ner-

vous and immune systems when nerve dam-

age occurs. After differentiation, the mRNA

and protein levels of the purine receptor

P2X7 increased significantly in ADSCs, and

inhibiting the P2X7 receptor could prevent

ATP-induced cell death

. However, in this

study, the expression of P2X7 in uADSCs and

dADSCs was not significantly different. This

may suggest that P2X7 plays a fundamental

role in maintaining the proliferation and dif-

Differentiation of adipose-derived stem cells enhance secretion of neurotrophic factors 277

Vol. 64(3): 267 - 280, 2023

ferentiation of uADSCs and dADSCs under

physiological conditions.

In conclusion, there are no differenc-

es in myelin production in the two groups

studied, despite the increased neurotrophic

factors and their receptors in the dADSCs

group being more potent inducers of axonal

growth potential than uADSCs. dADSCs and

SCs are similar in morphology and function

in vitro and in vivo and are readily implanted

and proliferate rapidly

. Nevertheless, the

undifferentiated state of ADSCs enables

multiple-lineage differentiation and the es-

tablishment of a favorable environment for

nerve regeneration. uADSCs are easier to

obtain, have shorter incubation periods,

and are less costly than dADSCs, suggesting

their potential for nerve regeneration. Fur-

ther studies are needed to assess the poten-

tial of uADSCs and dADSCs.

Funding

This study was supported by the founda-

tion of key laboratory of Chinese medicine

innovation and transformation in Gansu

Province/Chinese medicine product engi-

neering laboratory of Gansu Province (ZY-

FYZH-KJ-2016-004), Talent innovation and

entrepreneurship science and technology

projects of Lanzhou city (2015-RC-20), and

Natural Science Foundation of Gansu Prov-

ince (21JR7RA453).

Conflict of interests

All authors declare that they have no

known competing financial interests or per-

sonal relationships that could have appeared

to influence the work reported in this paper.

Author’s ORCID numbers

• Xin Zeng:

0009-0007-9781-2943

• Yun He:

0009-0004-9783-629X

• Ya-nan Liu:

0009-0003-4550-7446

• Fang Li:

0009-0008-5094-9763

• Zhen Li:

0009-0009-9519-0663

• Shu-yuan Zhang:

0009-0005-7689-0606

• Jing Gu:

0009-0003-6085-3574

• Li Lu:

0000-0002-2224-6963

Author contributions

XZ: conceptualization, methodology,

software, investigation, formal analysis, writ-

ing-original draft; YNL: conceptualization,

methodology, formal analysis, writing-origi-

nal draft; investigation; ZL: resources, writ-

ing-original draft; YH: conceptualization, vi-

sualization; FL: resources, supervision; SYZ:

software, validation; JG: resources, data cu-

ration, visualization; LL: conceptualization,

funding acquisition, supervision, writing -

review & editing.

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