The Extracts of Human Fetal Brain Induce the Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Dopaminergic Neuron Containing Cells

Abstract

Mesenchymal stem cells (MSCs) have the potential to differentiate into neuron-like cells, which may provide a new strategy for the clinical treatment of neurodegenerative diseases such as Parkinson's disease (PD). However, the application of MSCs in the patients is still limited as the reason of efficiency and safety of transplantation. The aim of this study is to develop a new method and induce human umbilical cord MSCs (hUCMSCs) into neuron-like cells. Results from flow cytometry indicate that the isolated MSCs from hUCMSCs exhibited a typical phenotype of adult stem cells and express CD44, CD54, CD73, CD90, CD105, CD166, and HLA-ABC. Furthermore, the induced cells from hUCMSCs could spontaneously express different neural cell markers [neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP)], even transcription factors related to dopaminergic neuron's development (Nurr1, Wnt-1, and En-1). Moreover, after treatment of EHFBT (extracts of human fetal brain tissue), hUCMSCs can express neuronal markers such as Nestin, LIM homeobox transcription factor 1 beta (LMX1B), dopamine beta hydroxylase (DBH), and dopamine transporter (DAT). In summary, a method that can induce hUCMSCs into dopaminergic neuron containing cells is established in vitro by the treatment of EHFBT. This would provide us a new cell source for PD in clinical treatment in the future.

Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by degeneration of dopaminergic neurons in the substantia nigra. To date, there is still no ideal therapy for PD (Jaime et al., 2018). Dopaminergic drugs have been studied for many years, however, these drugs cannot prevent dopaminergic cell loss, so cellular transplantation, especially dopaminergic cells, is thought to hold great potential to deal with Parkinson’ disease. Stem cells are nowadays considered the best candidates as an alternative cell source of PD patients, but whether they can differentiate into dopaminergic cells in vivo after transplantation is still not clear.

In the search for a renewable source of dopamine-producing cells, human embryonic stem cells, human adult stem cells and induced pluripotent stem cells from PD patients are extensively studied. Human fetal brain tissue and neural stem cells/progenitors have been investigated previously (Barker et al., 2015; Freed et al., 2001; Kim et al., 2002; Lige and Zengmin, 2016). Recently, a number of studies have examined the ability of mesenchymal stem cells (MSCs), including human umbilical cord MSCs (hUCMSCs), to differentiate into dopamine-producing cells, reinnervate the striatum, and ameliorate behavioral deficits in Parkinsonian models due to their convenient source, safety, without ethical limitation, and immunomodulatory effects (Campeau et al., 2014; Mendes et al., 2018).

Neuronal differentiation of MSCs has been achieved through a wide range of approaches, including growth factors, signaling molecules, and chemicals. hUCMSCs can differentiate into neuron-like cells by different inducing methods, including (1) growth factors such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and beta nerve growth factor (BNGF); (2) antioxidants such as dimethyl sulfoxide (DMSO) and β-mercaptoethanol (BME); (3) gene transfection; and (4) coculture, providing a good prospect for clinical applications (Sanchez-Ramos et al., 2000; Sanchez-Ramos, 2002). The extracts of human fetal brain tissue (EHFBT) were obtained from some brain tissues of the aborted human fetus, homogenated and centrifuged; the supernatant extracts were stored as EHFBT.

Some studies have demonstrated that tissue extracts from different parts of the human body can induce MSCs to differentiate into osteoblasts (Chevallier et al., 2010), adipocytes (Ge et al., 2009), and myocardial cells (Ma et al., 2011; Perán et al., 2010; Sarkanen et al., 2012) in vitro. However, the role of EHFBT in the induction of MSCs into neural-like cells is still unknown, especially dopaminergic neurons. In this study, we show that hUCMSCs can spontaneously express neural cell markers [NSE (neuron-specific enolase) and GFAP (glial fibrillary acidic protein)], and dopaminergic neuron factors such as Nurr1, Wnt-1, and En-1. The expression of neuron markers NSE, DAT (dopamine transporter), DBH (dopamine beta hydroxylase), and TH (tyrosine hydroxylase) was also detected in the induced cells after EHFBT treatment. Taken together, out results show that EHFBT can induce hUCMSCs into dopaminergic neuron containing cells and they would be a possible cell source for the treatment of PD in the future.

Materials and Methods

Isolation and culture of hUCMSCs

Human umbilical cords obtained from women with full-term pregnancies whose babies were delivered by cesarean section were provided by the Third Affiliated Hospital of Xinxiang Medical University, China. This study was approved by the Ethics Committee of Xinxiang Medical University (XYLL-2016010).

The collected umbilical cords were placed in high-glucose Dulbecco's modified Eagle's medium (DMEM)/F12 culture medium and then were rinsed thoroughly with D-Hank's medium. After withdrawing the blood sample, the umbilical artery and umbilical vein were removed. The tissue was cut into 1 mm³ sections, digested with 0.2% collagenase II, and then placed into a culture flask containing 2 ng/mL EGF, 20% fetal bovine serum (FBS), 25 mM L-glutamine, and 100 U/mL penicillin and 100 mg/mL streptomycin in high-glucose DMEM/F12. When the cells achieved 80%–90% confluency, they were passaged at a 1:3 ratio and cultured in high-glucose DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. The flasks were placed into an incubator at 37°C with 5% CO₂.

Preparation of EHFBT

All the procedures for preparation of EHFBT were carried out in accordance with the approval by the Ethics Committee of Xinxiang Medical University, the fetus was inevitably aborted, and the fetal brain used in this study was under the family members' approval.

The fetus was rinsed by alcohol at first, washed with 0.01 M phosphate-buffered saline (PBS) on ice, and homogenated in low-glucose DMEM (1 g tissue in 500 μL low-glucose DMEM). Then the samples were centrifuged at 12,000 r/min for 30 minutes at 4°C. Finally, the supernatant extracts were filtered with a 0.22 μm filter, and the protein content was measured and adjusted to 10 mg/mL with low-glucose DMEM for further use.

Differentiation of hUCMSCs into neural cells

To analyze whether hUCMSCs can differentiate into neuron-like cells, third-generation hUCMSCs were used for the induction. The cells were plated into a 6-well plate at 0.5 × 10⁵ cells per well, 2 mL low-glucose DMEM with 10% FBS was added for culture, left till the cells' confluence reached at about 60%–70%, then 20% (v/v) EHFBT was added to the culture medium for 3 to 5 days, and then, the culture medium was changed to DMEM with 10% FBS for continued culturing.

RNA extraction and real-time PCR analysis

RT-PCR was used to detect the expression of neural cell markers. Total RNA of the control hUCMSCs and the EHFBT-treated hUCMSCs was extracted using the RNA Extraction Kit for mammalian cells. Then the first-strand cDNA was synthesized from total RNA using the First Strand cDNA Synthesis Kit according to the manufacturer's instructions.

RT-PCR was run as follows: 95°C 3 minutes for predenaturation, 30 cycles at 95°C 30 seconds, 58°C–67°C 30 seconds, and 72°C 30–55 seconds, and 72°C 5 minutes for the final elongation, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as an internal control. Amplified DNA fragments were electrophoresed using 1% agarose gel. The sequences of primers and annealing temperature used in this study are shown in Table 1.

Table 1.

The Primers Used for mRNA Expression Analysis

Genes	Primer sequences	Product sizes (bp)	Tm (°C)
Nestin	For: AACAGCGACGGAGGTCTCTA	220	60.7
Nestin	Rev: TTCTCTTGTCCCGCAGACTT	220	60.7
NF(H)	For: TGAACACAGACGCTATGCGCTCAG	398	62
NF(H)	Rev: CACCTTTATGTGAGTGGACACAGAG	398	62
S100B	For: GGAAATCAAAGAGCAGGAGGT	254	60.3
S100B	Rev: ATTAGCTACAACACGGCTGGA	254	60.3
NSE	For: GTGTCTCTGGCCGTGTGTAA	371	62
NSE	Rev: GTGTAGCCAGCCTTGTCGAT	371	62
GFAP	For: GTCCATGTGGAGCTTGAC	406	60
GFAP	Rev: CATTGAGCAGGTCCTGGTAC	406	60
Nurr1	For: CGATGCCTTGTGTTCAGGCGCAG	858	67
Nurr1	Rev: AGCCTTTGCAGCCCTCACAGGTG	858	67
Wnt-1	For: CGGGCAACAACCAAAGTCG	321	60
Wnt-1	Rev: GACAGTTCCAGCGGCGATTC	321	60
GAPDH	For: CAAGGTCATCCATGACAACTTTG	496	58
GAPDH	Rev: GTCCACCACCCTGTTGCTGTAG	496	58

NF, neurofilament protein; NSE, neuron-specific enolase; GFAP, glial fibrillary acidic protein.

Western blotting analysis

To detect the protein expression levels of dopaminergic transcription factors and neuron makers, protein extracts were prepared from uninduced and induced hUCMSCs using the protein extraction kit for mammalian cells.

The cell lysates were electrophoresed (15 μg per lane) through polyacrylamide gels and electroblotted onto the polyvinylidene difluoride membrane. Blocked with 5% nonfat dry milk in TBST (tris-buffered saline with Tween 20) for 1 hour at room temperature, the primary antibodies (purchased from Santa Cruz) of mouse monoclonal antibodies Nurr1, NSE, GFAP, β-III-tubulin, and TH, and goat polyclonal antibodies of Wnt-1, En-1, and DAT were incubated at 1:1000 dilution overnight at 4°C followed by incubation with HRP-conjugated secondary antibodies (Zhongshan Golden Bridge). β-actin was used as an internal control and the protein of PC12 cells served as a positive control. Antibodies were detected using an enhanced chemiluminescence reagent.

Immunofluorescence staining

Cells plated on chamber slides were fixed in 4% PFA (paraformaldehyde) for 15 minutes at room temperature, washed with PBS, and permeabilized with 0.2% Triton X-100 in PBST for 20 minutes, after blocking with 5% FBS for 1 hour. Cells were incubated overnight in a primary antibody dilution buffer containing the following primary antibodies separately: NSE (1:250), GFAP (1:250), Nestin (1:250), DAT (1:250), DBH (1:250), and LMX1B (LIM homeobox transcription factor 1 beta) (1:250). The next day, the cells were rinsed with PBS and secondary antibodies were added to incubate for 1–2 hours at room temperature in the dark, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Images were acquired using the Nikon fluorescence microscope.

Statistical analysis

Data were analyzed with a two-tailed Student's t-test, and a value of p < 0.05 was considered significant, *p < 0.05, **p < 0.005, and ***p < 0.001. Values are given as mean ± SD (standard deviation). Also, the ImageJ software is used for density analysis.

Results

Analysis of the cellular phenotype of hUCMSCs

After culturing for 3–5 days, many spindle fibroblast-like morphology cells grow out around the tissue section. When the confluence of the cells reached about 80%–90%, the tissue sections were removed and the cells were passaged. The phenotype of the cells (Passage 3, P3) was identified using a flow cytometer. The flow cytometry analysis results in Figure 1 show that they positively express adult stem cell markers CD44, CD54, CD73, CD90, CD105, CD116 and HLA-ABC; no obvious expression of hematopoietic cell marker CD45, hematopoietic stem cell marker CD34, type II antigen presentation molecular HLA-DR, or immune cell-related markers CD80 and CD86. These results show that the isolated cells are MSCs, but not hematopoietic cells. To get good experimental results, hUCMSCs within P4 were used for the following experiments.

FIG. 1.

Flow cytometer analysis of hUCMSCs. To determine the immunophenotype of hUCMSCs, the cells at passages P3 were stained by corresponding conjugated antibodies and analyzed by flow cytometry analysis. The P3 hUCMSCs positively expressed classical MSC markers (CD44, CD54, CD73, CD90, CD105, and CD116) and HLA-ABC; negatively expressed hematopoietic stem cell markers (CD34 and CD45), T cell markers (CD80 and CD86) and HLA-DR. FACS; hUCMSCs, human umbilical cord mesenchymal stem cells. Color images are available online.

hUCMSCs express neural cell markers

After 2 weeks of culturing, the expression of neuron- or dopaminergic neuron-specific markers of the induced hUCMSCs was detected. RT-PCR assay demonstrated that the induced hUCMSCs can express neuron markers Nestin, NSE, and NF (neurofilament protein), astrocyte markers GFAP and S100B, dopaminergic neuron phenotypic marker Nurr1 and dopaminergic neuron-specific transcription factor Wnt-1 (Prakash and Wurst, 2006), and pro-DA neurogenesis effectors En-1 at the same time, but different mRNA expression levels (Fig. 1A). Furthermore, results of Western blotting confirmed that hUCMSCs can express neuron markers after a period of culture at the protein level (Fig. 2B, C). These results suggest that hUCMSCs have the potential to differentiate into neural-like cells mainly containing dopaminergic neuron-like cells and astrocytes.

FIG. 2.

hUCMSCs express neural cell markers. (A) RT-PCR analyzed the expression of neural cell markers in hUCMSCs. From right to left: GAPDH, Nurr1, NSE, Nestin, NF, GFAP, Wnt-1, S100B, and En-1. (B, C) Western blotting analyzed protein expression of the glial cell marker GFAP, neural cell markers and NSE, and dopaminergic neuron-related transcription factors (Nurr1, Wnt-1, and En-1) in hUCMSCs at 2 and 4 weeks. p Values are determined by Student's t test. Error bars indicate mean ± S.D. *p<0.05. NF, neurofilament protein; NSE, neuron-specific enolase; GFAP, glial fibrillary acid protein. Color images are available online.

Morphological changes of hUCMSCs during induction by EHFBT

To obtain the dopaminergic neuron-like cells, hUCMSCs were treated with 20% EHFBT in culture medium. The morphology of hUCMSCs changed obviously during culture with EHFBT containing medium. After 3 days of treatment by EHFBT, some of the cell bodies showed a typical neuronal morphology. After induction with EHFBT for 5 days, the differentiated hUCMSCs changed to a spindle and spherical shape. The induced hUCMSCs can be cultured in a normal complete medium for up to 50 days after induction by EHFBT, the synapses of cells elongated, and Nissl bodies were observed obviously (Fig. 3A–D).

FIG. 3.

Morphological changes of hUCMSCs during induction by EHFBT. (A) Undifferentiated hUCMSCs; (B) hUCMSCs treated by 20% EHFBT for 3 days; (C) hUCMSCs treated with 20% EHFBT for 5 days; (D) hUCMSCs in normal medium for 50 days after induction with EHFBT for 5 days. Scale bar: 100 μm. EHFBT, extracts of human fetal brain tissue.

The neural markers of hUCMSCs during induction by EHFBT

To test the changes of neuronal markers during induction, immunofluorescence and Western blotting were performed. The results indicate that the positive expression rates of NSE, GFAP, Nestin, LMX1B, DBH, and DAT are 18.32% ± 4.07%, 16.94% ± 7.56%, 14.47% ± 3.20%, 7.17% ± 3.28%, 1.81% ± 1.07%, and 5.65% ± 0.93%, respectively, after induction for 3 days, and the positive expression rates changed to 22.08% ± 2.75%, 17.83% ± 5.55%, 23.82% ± 6.05%, 11.98% ± 2.71%, 11.83% ± 2.75%, and 13.86% ± 5.50%, respectively, after induction for 5 days in differentiated hUCMSCs, respectively (Fig. 4A–D). Meanwhile, the results of Western blotting showed that the expression of NSE, GFAP, DAT, and TH increased significantly during induction from days 3 to 5. However, β-III-tubulin, one of the earlier neuronal markers, downregulated on day 5 compared with day 3 (Fig. 5A, B).

FIG. 4.

Immunofluorescence assay of neural cell markers during induction by EHFBT. (A–D) Immunofluorescence staining showed that the expression of neuron markers NSE, GFAP, and dopaminergic neuron markers LMX1B, DAT, Nestin, and DBH in hUCMSCs after induction by EHFBT for 3 and 5 days. Scale bar:100 μm. p Values are determined by Student's t test. Error bars indicate mean ± S.D. DAT, dopamine transporter; DBH, dopamine beta hydroxylase; LMX1B, LIM, homeobox transcription factor 1 beta. Color images are available online.

FIG. 5.

Western blotting assay of neural cell markers during induction by EHFBT. (A, B) Western blotting analyzed neural cell markers NSE, GFAP, β-III-tubulin, and DAT and TH expression in hUCMSCs after induction by EHFBT for 3 and 5 days. p Values are determined by Student's t test. Error bars indicate mean ± S.D. *p<0.05; **p<0.005; ***p<0.001. TH, tyrosine hydroxylase. Color images are available online.

Discussion

MSCs are stem cells derived from the mesoderm and can be isolated from many tissues such as bone marrow, fat, placenta, blood, and the umbilical cord. They have the potential of self-renewal, as well as a high proliferative and multidirectional differentiation potential. MSCs can differentiate into the same kind of cells that are derived from the mesoderm, and can also transdifferentiate into cells such as those derived from the ectoderm. Compared with stem cells derived from bone marrow, placenta, and other adult tissues, hUCMSCs have greater practical advantages in clinical treatments (Gazdic et al., 2015; Hashemian et al., 2015; Liu et al., 2018; Patel et al., 2008; Uccelli et al., 2008; Undale et al., 2009) and have evoked many scientists' interests.

Recently, a lot of reports demonstrated that MSCs can differentiate into neuron-like cells by different kinds of induction methods (Cho et al., 2008; Kim et al., 2011; Sun et al., 2010), which may bring new hope to people in clinical treatment and regenerative medicine of neurodegenerative diseases (Ende and Chen, 2001; Ende et al., 2001). The induction methods using neurotrophic factors (Choong et al., 2007) and toxic chemicals, such as DMSO or β-mercaptoethanol (Liu et al., 2005), were not safe enough for clinical use and encountered many limitations and restrictions in clinical application.

In our study, EHFBT was used to induce the hUCMSCs to differentiate into neuron-like cells. The EHFBT contains many kinds of growth factors and neurotrophic factors and can mimic the special human brain microenvironment, playing a vital role for the treatment of central nervous system disorders such as PD, Alzheimer's disease, and Huntington's disease (Guiney et al., 2017; Mi et al., 2019; Morris et al., 2018). The effect of brain tissue homogenate may be related to the multiple nerve growth factors contained in it. Chopp found that a variety of nerve cell growth factors such as brain-derived neurotrophic factor, nerve growth factor, bFGF, and glial-derived neurotrophic factors may play key roles in this differentiation process (Chen et al., 2002),

Furthermore, we found that NSE and GFAP were expressed at nearly equal levels in treated and untreated hUCMSCs with EHFBT, which is consistent with the data that neuron-like cells were induced from bone marrow MSCs by a cocktail of DMSO, BHA (butyl hydroxyanisole), KCl, valproic acid, forskolin, hydrocortisone, and insulin, respectively (Chevallier et al., 2010; Woodbury et al., 2000).

Our results demonstrate that the hUCMSCs can differentiate into neuron-like cells by EHFBT induction and provide evidences that the hUCMSCs may have the ability to differentiate into neuron-like cells when transplanted into the brain. However, our data show that hUCMSCs can express neural cell markers NSE, GFAP, S100B, Nurr1, Wnt-1, and En-1 in normal culture medium. Some articles have reported similar results in bone marrow-derived MSCs (Tondreau et al., 2004; Tseng et al., 2007). These results demonstrated that hUCMSCs have the tendency to differentiate into neuron-like cells under normal condition.

Our study initially confirmed that the EHFBT can induce hUCMSCs to express the neuronal marker, and has the ability to induce hUCMSCs to differentiate into neuron-like cells (mainly neurons, a small portion of glial cells contained), the differentiated cells are not a single cell type of nervous system cells, and this may explain the β-III-tubulin protein expression level change on day 5 than day 3, the expression pattern of β-III-tubulin and other neuron markers in hUCMSC may be very complicated than the data had shown when induced by the tissue extracts in vitro, and whether they have the corresponding neuronal cell functions needs to be further studied.

Also, how the brain tissue homogenate exerts its differentiation-inducing effect may be closely related to its concentration, action time, and the specific environment in which it works, and how to induce hUCMSCs to differentiate into neuron-like cells in vivo is more complicated than in vitro.

Footnotes

Author Disclosure Statement

The authors declare they have no conflicting financial interests.

Funding Information

This study was supported by the Project of Science and Technology Department of Henan Province (162102310493 and 162102210117), the Key Project of Science and Technology Research of Henan Provincial Education Department (18A180029 and 19A180027), the National Natural Science Foundation of China (81671619 and U1804186), and the National Undergraduate Training Programs for Innovation and Entrepreneurship (201510472013).

References

Barker

R.A.

, Drouin-Ouellet

, and Parmar

(2015). Cell-based therapies for Parkinson disease—Past insights and future potential. Nat. Rev. Neurol. 11, 492–503.

Campeau

, Soler

, Sittadjody

, Pareta

, Nomiya

, Zarifpour

, Opara

E.C.

, Yoo

J.J.

, and Andersson

K.E.

(2014). Effects of allogeneic bone marrow derived mesenchymal stromal cell therapy on voiding function in a rat model of Parkinson disease. J. Urol. 191, 850–859.

Chen

, Katakowski

, Li

, Lu

, Wang

, Zhang

, Chen

, Xu

, Gautam

, Mahmood

, and Chopp

(2002). Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts: Growth factor production. J. Neurosci. Res. 69, 687–691.

Chevallier

, Anagnostou

, Zilber

, Bodivit

, Maurin

, Barrault

, Bierling

, Hernigou

, and Layrolle

, Rouard

(2010). Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate. Biomaterials, 31, 270–278.

Cho

M.S

, Hwang

D.Y.

, and Kim

D.W.

(2008). Efficient derivation of functional dopaminergic neurons from human embryonic stem cells on a large scale. Nat. Protoc. 3, 1888–1894.

Choong

P.F.

, Mok

P.L.

, Cheong

S.K.

, Leong

C.F.

, and Then

K.Y.

(2007). Generating neuron-like cells from BM-derived mesenchymal stromal cells in vitro. Cytotherapy, 9, 170–183.

Ende

, and Chen

(2001). Human umbilical cord blood cells ameliorate Huntington's disease in transgenic mice. J. Med. 32, 231–240.

Ende

, Chen

, and Ende-Harris

(2001). Human umbilical cord blood cells ameliorate Alzheimer's disease in transgenic mice. J. Med. 32, 241–247.

Freed

C.R.

, Greene

P.E.

, Breeze

R.E.

, Tsai

W.Y.

, DuMouchel

, Kao

, Dillon

, Winfield

, Culver

, and Trojanowski

J.Q.

(2001). Transplantation of embryonic dopamine neurons for severe Parkinson's disease. N. Engl. J. Med, 344, 710–719.

10.

Gazdic

, Volarevic

, Arsenijevic

, and Stojkovic

(2015). Mesenchymal stem cells: A friend or foe in immune-mediated diseases. Stem Cell Rev. 11, 280–287.

11.

, Liu

, Li

, Wu

, Tu

, Shi

, and Chen

(2009). Chemical and physical stimuli induce cardiomyocyte differentiation from stem cells. Biochem. Biophys. Res. Commun. 381, 317–321.

12.

Guiney

S.J.

, Adlard

P.A.

, Bush

A.I.

, Finkelstein

D.I.

, and Ayton

(2017). Ferroptosis and cell death mechanisms in Parkinson's disease. Neurochem. Int. 104, 34–48.

13.

Hashemian

S.J.

, Kouhnavard

, Nasli-Esfahani

(2015). Mesenchymal stem cells: Rising concerns over their application in treatment of type one diabetes mellitus. J. Diabetes Res. 2015, 675103.

14.

Jaime

, Lais

, and Susan

H.F.

(2018). Update in therapeutic strategies for Parkinson's disease. Curr. Opin. Neurol. 31, 439–447.

15.

Kim

J.H.

, Auerbach

J.M.

, Rodríguez-Gómez

J.A.

, Velasco

, Gavin

, Lumelsky

, Lee

S.H.

, Nguyen

, Sánchez-Pernaute

, Bankiewicz

, and McKay

(2002). Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’ disease. Nature, 418, 50–56.

16.

Kim

E.S.

, Kim

G.H.

, Kang

M.L.

, Kang

Y.M.

, Kang

K.N.

, Hwang

K.C.

, Min

B.H.

, Kim

J.H

, and Kim

M.S.

(2011). Potential induction of rat muscle-derived stem cells to neural-like cells by retinoic acid. J. Tissue Eng. Regen. Med. 5, 410–414.

17.

Lige

, and Zengmin

(2016). Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Turk. Neurosurg. 26, 378–383.

18.

Liu

T.Y.

, Zhang

, Xiao

L.L.

, Yao

X.L.

, Feng

S.W.

, and Zeng

(2005). Induced differentiation of human cord blood monocytes into neuron-like cells in vitro. J. First Mil. Med. Univ. 25, 152–155.

19.

Liu

, Niu

, Yang

, Yan

, Liang

, Sun

, Shen

, and Lin

(2018). Biological characteristics of human menstrual blood-derived endometrial stem cells. J. Cell Mol. Med. 22, 1627–1639.

20.

, Blesch

, and Tuszynski

M.H.

(2004). Induction of bone marrow stromal cells to neurons: Differentiation, transdifferentiation, or artifact? J. Neurosci. Res. 77, 174–191.

21.

, Fox

, Shi

, Shen

, Liu

, Pappas

J.D.

, Cheng

, and Qu

(2011). Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells. Neurol. Res. 33, 1083–1093.

22.

Mendes

F.D.

, Ribeiro

P.D.C.

, Oliveira

L.F.

, de Paula

D.R.M.

, Capuano

, de Assunção

T.S.F.

, and da

Silva

. V.J.D. (2018). Therapy with mesenchymal stem cells in Parkinson disease: History and perspectives. Neurologist, 23, 141–147.

23.

, Gao

, Xu

, Cui

, Zhang

, and Gou

(2019). The emerging roles of Ferroptosis in Huntington's disease. Neuromol. Med. 21, 110–119.

24.

Morris

, Berk

, Carvalho

A.F.

, Maes

, Walker

A.J.

, and Puri

B.K.

(2018). Why should neuroscientists worry about iron? The emerging role of Ferroptosis in the pathophysiology of neuroprogressive diseases. Behav. Brain Res. 341, 154–175.

25.

Patel

A.N.

, Park

, Kuzman

, Benetti

, Silva

F.J.

, and Allickson

J.G.

(2008). Multipotent menstrual blood stromal stem cells: Isolation, characterization, and differentiation. Cell Transplant. 17, 303–311.

26.

Perán

, Marchal

J.A.

, López

, Jiménez-Navarro

, Boulaiz

, Rodríguez-Serrano

, Carrillo

, Sánchez-Espin

, de Teresa

, Tosh

, and Aranega

(2010). Human cardiac tissue induces transdifferentiation of adult stem cells towards cardiomyocytes. Cytotherapy, 12, 332–337.

27.

Prakash

, and Wurst

. (2006) Genetic networks controlling the development of midbrain dopaminergic neurons. J. Physiol. 575(Pt 2), 403–410.

28.

Sanchez-Ramos

, Song

, Cardozo-Pelaez

, Hazzi

, Stedeford

, Willing

, Freeman

T.B.

, Saporta

, Janssen

, Patel

, Cooper

D.R.

, and Sanberg

P.R.

(2000). Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp. Neurol. 164, 247–256.

29.

Sanchez-Ramos

JR.

(2002). Neural cells derived from adult bone marrow and umbilical cord blood. J. Neurosci. Res. 69, 880–893.

30.

Sarkanen

J.R.

, Kaila

, Mannerström

, Räty

, Kuokkanen

, Miettinen

, and Ylikomi

(2012). Human adipose tissue extract induces angiogenesis and adipogenesis in vitro. Tissue Eng. Part A, 18, 17–25.

31.

Sun

, Allison

, McLaughlin

, Sledge

, Waters-Pick

, Wease

, and Kurtzberg

(2010). Differences in quality between privately and publicly banked umbilical cord blood units: A pilot study of autologous cord blood infusion in children with acquired neurologic disorders. Transfusion, 50, 1980–1987.

32.

Tondreau

, Lagneaux

, Dejeneffe

, Massy

, Mortier

, Delforge

, and Bron

(2004). Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation. Differentiation, 72, 319–326.

33.

Tseng

P.Y.

, Chen

C.J.

, Sheu

C.C.

, Yu

C.W.

, and Huang

Y.S.

(2007). Spontaneous differentiation of adult rat marrow stromal cells in a long-term cultures. J. Vet. Med. Sci. 62, 95–102.

34.

Uccelli

, Moretta

, and Pistoia

(2008). Mesenchymal stem cells in health and disease. Nat. Rev. Immunol. 8, 726–736.

35.

Undale

A.H.

, Westendorf

J.J.

, Yaszemski

M.J.

, and Khosla

(2009). Mesenchymal stem cells for bone repair and metabolic bone diseases. Mayo Clin. Proc. 84, 893–902.

36.

Woodbury

, Schwarz

E.J.

, Prockop

D.J.

, and Black

I.B.

(2000). Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364–370.