Capacity of human umbilical cord-derived mesenchymal stem cells to differentiate into sweat gland-like cells: a preclinical study

Abstract

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) possess various advantageous properties, including self-renewal, extended proliferation potential, multi-lineage differentiation potential and capacity for differentiating into sweat gland-like cells in certain conditions. However, little is known about the effect of clinical-grade culture conditions on these properties and on the differentiative potential of hUC-MSCs. In this study, we sought to investigate the properties of hUC-MSCs expanded with animal serum free culture media (ASFCM) in order to determine their potential for differentiation into sweat gland-like cells. We found that primary cultures of hUC-MSCs could be established with ASFCM. Moreover, cells cultured in ASFCM showed vigorous proliferation comparable to those of cells grown in classical culture conditions containing fetal bovine serum (FBS). Morphology of hUC-MSCs cultured in ASFCM was comparable to those of cells grown under classical culture conditions, and hUC-MSCs grown in both of the two culture conditions tested showed the typical antigen profile of MSCs—positive for CD29, CD44, CD90, and CD105, and negative for CD34 and CD45, as expected. Chromosomal aberration assay revealed that the cells were stable after long-term culture under both culture conditions. Like normal cultured MSCs, hUC-MSCs induced under ASFCM conditions exhibited expression of the same markers (CEA, CK14 and CK19) and developmental genes (EDA and EDAR) that are characteristic of normal sweat gland cells. Taken together, our findings indicate that the classical culture medium used to differentiate hUC-MSCs into sweat gland-like cells can be replaced safely by ASFCM for clinical purposes.

Keywords

umbilical cord / mesenchymal stem cells / sweat gland / preclinical

Introduction

Deep burn or trauma can cause serious damage to the normal structure and function of the skin and skin appendages. Damage to the structure and function of sweat glands significantly affects the quality of life of an individual. Several studies suggest that mesenchymal stem cells (MSCs) can participate effectively in promoting repair and regeneration of skin and sweat glands and may improve the quality of life of patients [1]. MSCs are an excellent candidate for cell therapy because of their various advantages such as ease of collection, short culture duration and low immunogenicity. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are a safe and accessible source for large quantities of stem cells in comparison to fetal MSCs and bone marrow-derived MSCs (BM-MSCs). Viability of umbilical cord as a stem cell source is supported by several studies that have demonstrated the presence of abundant MSCs in human umbilical cords [1-4].

As with BM-MSCs, previous studies have shown that hUC-MSCs can also be induced to differentiate into adipocytes, osteocytes, chondrocytes, neurons, endothelial cells, cardiomyocytes and skeletalmyocytes [4-11]. However, despite such exciting prospects, the effects of clinical-grade culture conditions on cell bioproperties and the differentiative potential of hUC-MSCs have not yet been investigated fully. In previous studies, we had successfully induced MSCs from both BM and hUC to differentiate into sweat gland-like cells [12-14]. Importantly, findings from these studies suggested that hUC-MSCs could be induced into sweat gland-like cells effectively under a novel and feasible system. This implied that hUC-MSCs could be a promising resource for sweat gland restoration after skin injury. Although reliable and validated methods for deriving, growing and preserving hUC-MSCs are within reach, the greatest impediment to the provision of hUC-MSCs for therapeutic purposes in sweat gland repair is the lack of comprehensive characterization of hUC-MSCs in clinical-grade culture conditions. In this study, we investigated the characteristics of hUC-MSCs expanded with animal serum free culture media (ASFCM) and evaluated their capacity to differentiate into sweat gland-like cells for future treatment of sweat gland injury.

Materials and methods

Collection of human umbilical cord and cell culture

Human umbilical cord samples were obtained from six patients who underwent routine accouchement procedures at the First Affiliated Hospital of Chinese PLA General Hospital and were donated with the patients’ informed consent. The process was reviewed and approved by the Institutional Review Board and the Ethics Committee of the First Affiliated Hospital of Chinese PLA General Hospital. Tissue was washed in Hank’s balanced salt solution (Gibco, USA) for 0.5-1 h at 4 °C. After removing umbilical cord blood vessels, the remaining cord tissue containing Wharton’s jelly was minced with a scalpel or surgical scissors and treated with collagenase type II (2 mg/ml; Sigma, USA) for 8-12 h at 37°C. Then, umbilical cord tissue was washed and the suspensions were centrifuged at 300 × g for 5 min at room temperature. Cells obtained in this manner were divided into two groups. Cells in group 1 were cultured in animal serum free culture media (ASFCM)(GIBCO^®, USA, STEMPRO MSC SFM CTS™) with 5 ng/ml vascular endothelial growth factor (VEGF; Gibco, USA), in an incubator (Termo, USA) with 5% CO₂ at 37 °C. The media was replaced every 3-4 days. Cells in group 2 were cultured in fresh basic medium containing Glucose Dulbecco’s modified Eagle’s medium (DMEM-F12) with 10% fetal bovine serum (FBS; Gibco, USA), 100 mg/ml penicillin, 100 mg/ml streptomycin, 1 mg/ml amphotericin B, 5 ng/ml vascular endothelial growth factor (VEGF; Gibco, USA), in an incubator (Termo, USA) with 5% CO₂ at 37 °C. The media was replaced every 2-3 days. When adherent cells reached 80%-90% confluence, they were harvested and inoculated into 25 cm² culture flasks (Costar, Corning, USA) at 1 × 10⁴ cells/ml, and cultured in an incubator with 5% CO₂ at 37 °C. Primary and passaged cultures were routinely observed under a phase-contrast inverted microscope (Leica, Germany) and verified to have no hematopoietic cells [7,11].

Preparation of sweat gland cell-conditioned medium

Small pieces (1 cm × 1 cm × 0.5 cm) of normal whole skin were received from three patients with their signed consent and the procedure was approved by the Research Ethics Committee of the First Affiliated Hospital of PLA General Hospital. After removing subcutaneous fat, the skin was minced into 0.5-1.0 mm³ pieces and incubated in collagenase type II for 4 h at 37 °C. Then, sweat gland cells (SGCs) were isolated from other tissue using needles under a ultraviolet-sterilized inverted phase-contrast microscope. A subset of SGCs were cultured in improved sweat gland cell-medium which consisted of ASFCM supplemented with 2 mM L-glutamine, insulin-transferring sodium selenite solution (1 ml/100 ml), 2 nM/ml triiodothyronine (T3), 0.4 mg/ml hemisuccinate hydrocortisone (Sigma) and 10 ng/ml human recombinant epidermal growth factor (EGF) (Sigma) (corresponding to group 1). The rest of the SGCs were cultured in the previously described [14] sweat gland cell-medium consisting of DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, insulin-transferring sodium selenite solution (1 ml/100 ml), 2 nM/ml triiodothyronine (T3), 0.4 mg/ml hemisuccinate hydrocortisone (Sigma) and 10 ng/ml human recombinant epidermal growth factor (EGF) (Sigma)] (corresponding to group 2). The medium was replaced every 3-4 days and the supernatants of antiquated media were collected. The supernatants were passed through a 2.2 mm diameter filter to eliminate bacteria and then preserved as a specific media for induction [12-14]. Normal SGCs were cultured in 3 ml of improved SGC medium at a density of 1×10⁴ cells/cm² as positive control for subsequent experiments.

Sweat gland cell differentiation of hUC-MSCs

A total of 1×10⁴ cells were cultured in improved SGC induction medium, which consisted of nine volumes of regular sweat gland cell medium (group 1) and one volume of heat-shocked sweat gland cell medium. The medium was replaced every 2-3 days for 3 weeks, during which the hUC-MSCs were observed through a microscope for morphological changes (induction group 1). Additionally, as a control, 1×10⁴ hUC-MSCs were cultured with our previous medium, which consisted of nine volumes of regular sweat gland cell medium (group 2) and one volume of heat-shocked sweat gland cell medium and the induction process was maintained for 3 weeks (induction group 2) [12-14]. All procedures in induction group 2 were conducted according to the technique described in our previous reports [12-14]. As a negative control, hUC-MSCs were cultured in basic medium (control group). Normal SGCs were also cultured as a positive control.

Flow cytometry analysis

Cell surface antigens were examined to characterize hUC-MSCs. A total of 3×10⁵-4.5×10⁵ cells were marked with the following anti-human antibodies and fluorescein isothiocyanate (FITC) secondary antibodies: CD29, CD34, CD44, CD45, CD90, CD105, CEA, CK14 and CK19 (Sigma) [14-16]. Labeled cells (5×10⁵) were obtained and analyzed (three replicates for each antigen) using a FACScan flow cytometer (Becton Dickinson, USA).

MTT assay

To compare the proliferation potency of different culture groups of hUC-MSCs, proliferation of cells cultured with basic medium, our previous conditioned induction medium (group 2) and improved conditioned induction medium (group 1) were measured with a tetrazolium-based colorimetric assay (MTT assay) [17]. A total of 1×10³ hUC-MSCs were plated into 96-well plates containing basic medium and two kinds of differentiation media (five replicates for each group). They were incubated for an additional 1, 2, 3 and 4 days. On days 1-4 of incubation, 20 μl 5 mg/ml MTT solution (Sigma) was added into each well and incubated for 4 h. The supernatants were removed and 200 μl dimethylsulphoxide (DMSO) solution were added. After shaking for 10 min, absorbance at 490 nm was measured (Spectra MR, Dynex, USA).

Karyotype analysis

At 10 passages, evaluation of telomere length and karyotype analysis were performed with technical assistance from the Department of Pathology (the First Affiliated Hospital of Chinese PLA General Hospital).

Reverse-transcription–polymerase chain reaction

Total RNA was isolated from normal SGCs, uninduced and induced hUC-MSCs of two culture conditions to examine the expression of anhidrotic ectodermal dysplasia (EDA) and EDA receptor (EDAR) based on the manufacturer’s instructions (TaKaRa, Japan). After induction, 1 × 10⁶ cells were harvested from each of the different groups. Transcript levels were determined following the manufacturer’s instructions. The following primers were used:

EDA: NM_001399.4, 156 bp

sense 5′-GGACGGCACCTACTTCATCTA-3′, antisense 5′-GCGGTATAGCAAGTGTTGTAGTT-3′

EDAR: NM_022336.3, 121 bp

sense 5′-CAGCCCGAGCGGAATACTC-3′, antisense 5′-CCGTAGCCACAGGACAGGTA-3′

β-actin: NM_001101.2, 285 bp

sense 5′-AGCGAGCATCCCCCAAAGTT-3′, antisense 5′-GGGCACGAAGGCTCATCATT-3′

Reverse-transcription-polymerase chain reaction (RT-PCR) was performed as described previously [14]. Briefly, the amplification step was run for 30-32 cycles after initial heating at 94 °C for 2 min, annealing for 30 s at 50-65 °C and extension for 10 min at 72 °C. The primers were used at a concentration of 0.05 mM in each reaction. The PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide (three replicates for each group). Quantitative analysis of RT-PCR bands was done using ImageJ software (http:// rsbweb. nih. gov/ij/; Research Services Branch, National Institute of Mental Health, Bethesda, MD, USA).

Immunoblotting analysis

Proteins were isolated from normal SGCs, uninduced and induced hUC-MSCs of two culture conditions, each containing the same amount (1 × 10⁶) of cells, in ice-cold RIPA buffer (containing 150 mM NaCl, 1.0% NP-40, 1% deoxycholate, 0.1% SDS, 20mM Tris, pH 7.5, and PMSF (Sigma)) and centrifuged (three replicates for each group). The pellets were then suspended in RIPA buffer+ 1% sodium dodecyl sulfate and sonicated to extract the less soluble proteins. Protein concentrations were measured using the Bradford method, using a Bio-Rad (USA) protein assay system. Extracted proteins were then denatured by adding β-mercaptoethanol (5% in final volume) and boiling for 5 min. A sample (40 mg) of each extract was added to a 12% Tris-glycine acrylamide gel (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was then blocked with 5% non-fat dried milk in 1×PBS containing 0.1% Tween-20 and incubated with primary antibodies overnight at 4°C. The following antibodies were used: mouse polyclonal anti-EDA (Abcam, USA) at a dilution of 4 mg/ml and rabbit polyclonal anti-EDAR (Santa Cruz Biotechnology, CA, USA) at a dilution of 1:200. After washing in PBS containing 0.1% Tween-20, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit antibodies (Santa Cruz Biotechnology) for 2 h at room temperature. The immunoreactive bands were then visualized using an enhanced chemiluminescence (ECL) kit (Amersham, Buckinghamshire, UK). All other procedures were carried out as described in our previous report [14].

Statistical analysis

All data are expressed as mean±standard error of the mean (SEM). Data were analyzed using the independent T- or Duncan’s Multiple Range Tests. Differences with P-values less than 0.05 were considered statistically significant.

Results

Morphology and phenotype of hUC-MSCs

Two groups of hUC-MSCs were observed through the microscope as spindle-shaped adherent cells from Wharton’s jelly in primary cultures (Fig.1-I). Flow cytometry analysis showed that the mean percentage of hUC-MSCs positive for mesenchymal markers CD29, CD44, CD90 and CD105 in group 1 were 95.68%, 99.22%, 94.34% and 99.46%, respectively, while those in group 2 were 94.74%, 98.54%, 95.17% and 99.23%, respectively. These cells were negative for the hematopoietic marker CD34 and CD45 in both groups (representative data from group 1 are shown in Supplemental Fig. S1). Thus, the characteristics of hUC-MSCs were highly similar to those of BM-MSCs.

Carcinoembryonic antigen (CEA) is a specific marker for SGCs in the adult skin, and both cytokeratin 14 (CK14) and cytokeratin 19 (CK19) are positive for normal SGCs [14,18,19]. Our results also revealed that uninduced hUC-MSCs were negative for these sweat gland markers in both groups (group 1: CEA, 0.77%; CK14, 1.58%. group 2: CEA, 0.89%; CK14, 1.65%). Representative data from group 1 are shown in Supplemental Fig.S1.

The mean percentages of each phenotype were compared between group 1 and group 2, and no significant difference was observed in the mean percentage of all markers (Fig. 1-II).

Proliferative potency of hUC-MSCs in different conditions

We examined the proliferation of hUC-MSCs in two induction groups while inducing them into sweat gland-like cells. Results of our MTT analysis revealed that the proliferative ability of hUC-MSCs in basic medium was maintained at a higher level than in induction groups 1 and 2 (*P<0.05). However, there was no significant difference between induction groups 1 and 2 (P>0.05). Thus, our results show that animal serum free culture condition (group 1) could maintain the same proliferation potency as our previously reported induction system (group 2) for differentiating hUC-MSCs into sweat gland-like cells (Fig. 2).

Sweat gland differentiation potency of hUCMSCs in different conditions

hUC-MSCs were cultured in both conditioned induction media in which they could differentiate into sweat gland-like cells and were observed for morphological changes under the microscope. In both of the two induction groups, hUC-MSCs began to lose their original morphology from week 1 (Fig.3A, induction group 1; Fig.3C, induction group 2), then appeared as normal sweat gland cells typically around 2.5-3 weeks (Fig.3B, induction group 1; Fig.3D, induction group 2).

Expression of sweat gland marker

After 3 weeks of induction, 1.5×10⁶ cells were harvested for expression of sweat gland markers (5 replicates in each group). Flow cytometry analysis showed the mean positive percentages of differentiated hUC-MSCs for CEA, CK14 and CK19 in group 1 were 86.3%, 61.4% and 84.2%, respectively. In comparison, in induction group 2, the mean percentages were 84.6%, 65.7% and 82.3%, respectively (Fig.4). This evidence implied that there was no significant difference between induction group 1 and 2 in differentiating hUC-MSCs into sweat gland-like cells (P>0.05).

Expression of EDA and EDAR

EDA and EDAR are proteins expressed in human SGCs, and mutations in them can cause deficiencies of the sweat glands [18]. Through quantitative comparison of areas from randomly selected RT-PCR bands, we found that both induction groups significantly expressed EDA and EDAR at nearly the same level (P>0.05) (Fig.5A, C). Through western blotting analysis, we also found that EDA and EDAR were expressed in induction groups 1 and 2 at similar levels (P>0.05, Fig.5B). Normal sweat glands and uninduced hUC-MSCs were used as positive and negative control, respectively. These results further illustrated that there was no significant difference between induction groups 1 and 2 in the extent of differentiation of hUC-MSCs into sweat gland-like cells.

Karyotype analysis

Five samples were investigated for chromosomal alternation by karyotype analysis at 10th passage of each induction group and no chromosomal alteration was noted (Fig. 6).

Discussion

Numerous studies have indicated that MSCs are multipotent, non-hematopoietic progenitor cells that are being explored as a promising new treatment for tissue regeneration. For example, MSCs have already been used in clinical trials as treatment for acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation [20,21] and for autoimmune diseases such as systemic sclerosis [22]. Our previous studies showed that MSCs could participate effectively in promoting regeneration of damaged sweat glands caused by deep burn or trauma. Repair of sweat glands could largely restore the deterioration of a survivor’s quality of life owing to the loss of sweat gland function. Among MSCs, hUC-MSCs have been considered as attractive candidates for regenerative medicine because these stem cells have faster proliferation and greater in vivo expansion capabilities compared with BM-MSCs. Thus, hUC-MSCs have been widely investigated in preclinical cell-based therapy studies as an alternative to BM transplantation. Therefore, the features and differentiative potential of these cells under clinical-grade conditions need to be carefully investigated before their release for clinical use.

In the present study, we monitored the features of long-term cultured hUC-MSCs under ASFCM in order to determine their differentiative potential into sweat gland-like cells. Our results showed that hUC-MSCs could be successfully cultured under ASFCM in vitro, without overt loss of their peculiar morphological and phenotypical characteristics. As expected, they maintained their typical spindle shape and constant growth rate. Importantly, we observed that hUC-MSCs cultured under ASFCM conditions maintained their differentiative potential into sweat gland-like cells as they did in previously described culture conditions [14] at levels comparable to those of normal cultured MSCs. EDA has been documented to be a key protein, and CEA, CK14 and CK19 are known to be key markers in the differentiation of sweat gland cells [23]. Like normal cultured MSCs, hUC-MSCs induced under ASFCM conditions exhibited expression of the same markers (CEA, CK14 and CK19) and developmental genes (EDA and EDAR) that are characteristic of normal sweat gland cells. Furthermore, karyotype analysis experiments at late passages revealed normal karyotype without expression of telomere maintenance mechanisms.

In conclusion, our data indicate that hUC-MSCs can be safely expanded in vitro and display similar behavior in maintaining their peculiar morphological, phenotypical and functional features under clinical-grade culture conditions compared with previously described culture conditions. Also, sweat gland differentiation potency of hUC-MSCs under these clinical-grade culture conditions was no less than that in previously reported culture conditions. Moreover, this study is the first to report the biosafety characteristics of sweat gland-like cells induced from hUC-MSCs under clinical-grade culture conditions. Our results provide support to the concept that the biological properties of hUC-MSCs remain suitable under clinical-grade culture conditions and could be used for clinical sweat gland repair to improve the quality of life of patients.

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