JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH J Tissue Eng Regen Med (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1914
ARTICLE
Skeletal myogenic differentiation of human urinederived cells as a potential source for skeletal muscle regeneration Wei Chen1,2, Minkai Xie1,3,4, Bin Yang1,5, Shantaram Bharadwaj1, Lujie Song1,3,4, Guihua Liu1, Shanhong Yi2, Gang Ye2, Anthony Atala1 and Yuanyuan Zhang1* 1
Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA Department of Urology, Xinqiao Hospital, Third Military Medical University, Chongqing, People’s Republic of China 3 Department of Urology, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, People’s Republic of China 4 Shanghai Oriental Institute for Urologic Reconstruction, Shanghai, People’s Republic of China 5 Department of Urology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China 2
Abstract Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25–55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from ‘rice-grain’-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Received 10 June 2013; Revised 11 March 2014; Accepted 20 April 2014
Keywords urine-derived stem cells; myogenic regeneration; tissue engineering; stem cells; differentiation; cell therapy
1. Introduction Skeletal muscle is one of the major tissues, comprising 40–50% of the human body by weight (Huard et al., 2002). Skeletal muscle has a robust capacity to repair itself, based on the satellite cells that are localized between the muscle fibre sarcolemma and the surrounding *Correspondence to: Yuanyuan Zhang, Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 391 Technology Way, Winston-Salem, NC 27157, USA. E-mail: [email protected] Copyright © 2014 John Wiley & Sons, Ltd.
basal lamina (Cossu and Biressi, 2005; Relaix and Zammit, 2012). However, when there is loss of volumetric muscle, as in congenital muscle dystrophy or severe muscle trauma, scar tissue will accumulate because of a lack of satellite cells, leading to a myogenic dysfunction (Grogan et al., 2011; Moyer and Wagner, 2011). Autologous adult stem cell injection therapy has provided a promising alternative for skeletal muscle regeneration. Progenitor cells or stem cells obtained from skeletal muscle (Liadaki et al., 2012; Mitchell et al., 2010), bone marrow (de la Garza-Rodea et al., 2011; Merritt
W. Chen et al.
et al., 2010), umbilical cord (Grabowska et al., 2012), adipose tissues (Goudenege et al., 2009; Mizuno, 2010), amniotic fluid (Ma et al., 2012) and, more recently, induced pluripotent cells (iPS) (Darabi et al., 2012) are regarded as possible candidates for use in this therapy. However, harvesting these types of cells is invasive and may cause complications and donor-site morbidity. An autologous stem cell source that can be obtained using non-invasive techniques would be highly desirable. Recently, we demonstrated that a subpopulation of urine-derived cells, termed urine-derived stem cells (USCs), possess stem cell capabilities, such as selfrenewal, paracrine effect, immunomodulatory properties and multipotential differentiation. These cells can differentiate into mesodermal cell lineages, such as osteocytes, chondrocytes, adipocytes, endothelial cells and myocytes, including smooth muscle cell differentiation and endodermal lineages (e.g. urothelial cells) (Bharadwaj et al., 2013; Lang et al., 2013; Liu et al., 2013; Wu et al., 2011; Zhang et al., 2008). These cells maintain higher telomerase activity and possess long telomeres; further, they retain a normal karyotype in culture medium containing epidermal growth factors, even after several passages. Importantly, these cells do not form teratomas in vivo. USCs express cell surface markers associated with pericytes and mesenchymal stem cells (Bharadwaj et al., 2011; Bodin et al., 2010; Zhang et al., 2008). These cells can be isolated from regular voided urine (Zhang et al., 2008) from each individual via a noninvasive, simple and low-cost approach. The USCs isolated from one single urine specimen can generate 50–100 million cells at early passage (P4) in 3–4 weeks, sufficient numbers to use for cell therapy (Zhang et al., 2008). Thus, the goal of this study was to assess whether USCs, obtained though simple, non-invasive procedures, can differentiate into skeletal muscle cells (Sk-MCs) and potentially be used to treat skeletal muscle regeneration.
70–80% confluence, the cells were trypsinized and transferred into six-well dishes (P1). When the cultures reached 70–80% confluence once more, the cells were transferred to a 100 mm culture dish for expansion. Cell clones (P3) were selected for testing the mesenchymal/ pericyte surface markers with flow cytometry (FCM). The USCs clones were analysed with MSC markers (CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133 and STRO-1), haematopoietic lineage markers (CD14,CD34,CD45), a pericyte marker (CD146), an embryonic stem cell marker (SSEA-4) and an endothelialrelated lineage marker (CD31) (Table 1). Human Sk-MCs as control were isolated from healthy gracilis muscles discarded from surgery, as previously described (Liu et al., 2013). Briefly, the muscle samples were minced into small pieces, then incubated in collagenase II (0.1% w/v)–dispase (4 mg/ml) solution prepared in Dulbecco’s minimum essential medium (DMEM) for 1 h at 37 °C with constant shaking (60 rpm). The liberated cells were collected (400 × g) and suspended in DMEM containing 10% horse serum and plated into a six-well tissue culture dish. After 5 days in culture, the medium was changed to SkGM2 (Lonza Biologics, Portsmouth, NH, USA) containing 10% FBS at 37 °C in a 5% CO2 cell incubator.
2.2. Proliferation assay USCs were cultured at a density of 5000 cells/cm2, with medium changes every 2 days. Cells were trypsinized and manually counted in a haemocytometer when the cell density reached 70–80% confluence (generally 3–5 days). The population doubling (PD) and doubling times (DT) were calculated using the following formulae (Table 2):
Table 1. Antibodies used in this study and their dilutions
2. Materials and methods 2.1. Cell culture The protocol for obtaining human urine samples for this study was approved by the Wake Forest University Institutional Review Board. USCs were isolated and characterized from 15 individual urine samples collected from six healthy individuals aged 25–55 years, as previously described (Bodin et al., 2010; Zhang et al., 2008). Briefly, voided urine samples were centrifuged at 500 × g for 5 min and the supernatant was removed. The cell pellet was gently resuspended in medium comprising of keratinocyte serum-free medium (KSFM) and embryonic fibroblast medium (EFM), mixed at a ratio of 1:1, and 5% fetal bovine serum (FBS) and plated in 24-well plates at 1 × 104 cells/well. Individual clones appeared 4–7 days after implantation. Each clone was plated into a single well of a 24-well dish, and when the well reached Copyright © 2014 John Wiley & Sons, Ltd.
Primary antibody
Source/cat. no.
Antibody dilution
CD14-APC CD24-FITC CD29-PE CD31-FITC CD34-FITC CD44-PE CD45-APC CD73-PE CD90-APC CD105-PE CD117-PE CD133-PE CD146-PE STRO-1-FITC SSEA-4-PE MsIgG1-PE MsIgG1-FITC MsIgG1-APC Myf5 (C-20) MyoD (C-20) Myosin (G-4) Desmin (RD301) NuMA
R&D Systems/FAB3832A BD Biosciences/BD 560992 BD Biosciences/BD 556049 BD Biosciences/BD 560984 BD Biosciences/BD 348053 BD Biosciences/BD 550989 BD Biosciences/BD 340943 BD Biosciences/BD550257 BD Biosciences/BD 559869 BD Biosciences/BD 560839 BD Biosciences/BD 340867 Miltenyi Biotec/MACS 130080801 BD Biosciences/BD 550315 Biolegend /340105 BD Biosciences/BD 560128 BD Biosciences/BD 559320 BD Biosciences/BD 555909 BD Biosciences/BD 555751 Santa Cruz/sc302 Santa Cruz/sc304 Santa Cruz/sc6956 Santa Cruz/sc23879 Millipore/mab1281
1:20 1:10 1:10 1:10 1:10 1:10 1:40 1:40 1:10 1:40 1:10 1:20 1:10 1:40 1:10 1:10 1:10 1:10 1:100 1:100 1:100 1:100 1:100
J Tissue Eng Regen Med (2014) DOI: 10.1002/term
Skeletal myogenic differentiation of hUSCs Table 2. Proliferative ability of USC clones USC clone A2 D3 B3
Average doubling time (DT)
Total population doublings (PD)
Starting cell number
Ending cell number
24.9 ± 9.4 29.9 ± 10.3 20.7 ± 4.1
56.7 48.0 32.2
1 1 1
1.17 × 10 14 2.79 × 10 9 4.89 × 10
PD = ln(Nf/Ni)/ln(2) and DT = Ct/PD, where Nf is the final number of cells, Ni is the initial number of cells and Ct is the culture time.
2.3. FACS analysis USCs (P3) were trypsinized and washed in phosphatebuffered saline (PBS) containing 0.5% bovine serum albumin (BSA). One million cells in 100 ml PBS containing 3% BSA were incubated with fluorochome-conjugated antibodies (Table 1) for 30 min at 4 °C. The cells were washed again, resuspended in 100 ml PBS containing 0.5% BSA (three times) and passed through a 70 mm cell filter before flow analysis using a FACS Calibur machine (BD Biosciences, Franklin Lakes, NJ, USA).
2.4. Optimization of myogenic culture medium Several media and supplements have been suggested for stem cell differentiation to a skeletal muscle lineage (De Coppi et al., 2007; Zuk et al., 2002). In the present study, USCs were induced to differentiate into Sk-MCs using the four different media listed below: 1. HD differentiation medium: DMEM (2 mM L-glutamine), 10% FBS, 5% horse serum, 1% penicillin–streptomycin, 50 μM hydrocortisone, 0.1 μM dexamethasone (Gibco/BRL). USCs (p3) were plated at a density of 1 × 104 cells/cm2 ™ on plates coated with Matrigel (1:1 diluted with medium; Collaborative Biomedical Products) and exposed to HD medium. The media were changed every 3 days up to 2 weeks (HD2W) and 4 weeks (HD4W) (Zuk et al., 2002).
2. SK differentiation medium: Sk-MC growth medium (SkGM-2, Lonza), followed by differentiation medium [DMEM:F-12 (1:1), 2% horse serum, 1% penicillin– streptomycin (Gibco/BRL). USCs (P3) were seeded at a density of 1 × 104 cells/cm2 on plastic plates precoated with Matrigel (1:1 diluted with medium) and exposed to SkGM-2 for 1 week, followed by differentiation medium. The differentiation medium was changed every 3 days up to 2 weeks (SK2W) and 4 weeks (SK4W) (Stern et al., 2009). 3. 1% CEE differentiation medium: DMEM (2 mM L-glutamine), 10% horse serum, 1% penicillin–streptomycin, 1.0% chick embryo extract (CEE; Gibco/BRL), 3 μM 5′Aza 2′deoxy cytidine (5-azaC; Sigma-Aldrich). USCs (P3) were seeded at a density of 1 × 104 cells/cm2 on plates precoated with Matrigel (1:1 diluted with medium) in Copyright © 2014 John Wiley & Sons, Ltd.
Total time for expansion (days)
Number of passages
55 51 30
13 11 9
17
1% CEE medium without 5-azaC; 12 h after seeding, 5-azaC was added to the culture medium for 24 h of incubation, then incubation continued in complete medium lacking 5-azaC, with medium changes every 3 days up to 28 days (De Coppi et al., 2007). 4. Conditioned differentiation medium: spent media from primary cultured human Sk-MCs. Conditioned medium was prepared from differentiated human skeletal muscle cells cultured in DMEM:F-12 (1:1) containing 2% horse serum and 1% penicillin–streptomycin at a confluence of 60–80%. The spent medium (15–18 h) was filtered (0.2 μM ) and used immediately or stored at –80 °C until further use. The USCs were plated at 1 × 104 cells/cm 2 on Matrigel (1:1 diluted with medium) with conditioned medium and then the medium was changed every 3–4 days up to 4 weeks. After optimization of the four induced media, our results showed that HD and SK differentiation media could induce USCs in myogenic lineage gene and protein expression at higher levels than the other two media (data not shown). We therefore focused on both HD and SK differentiation media for further analysis.
2.5. RNA analysis To determine myogenic differentiation of USCs with realtime PCR, the mRNA was extracted with an RNA isolation kit (5 PRIMEs, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Total RNA (5 μg) was converted to cDNA in a reaction containing random primers, nucleotides and reverse transcriptase enzymes, using a high-capacity cDNA reverse-transcription kit (Applied Biosystems, Foster City, CA, USA). One-tenth of the cDNA was then used for real-time analysis, along with Taqman Universal PCR Master Mix and Taqman gene expression probes, according to the manufacturer’s instructions, using a 7300 Real-time PCR system (Applied Biosystems). The primer pairs used in this study are listed in Table 3. To normalize mRNA levels, the GAPDH housekeeping gene was amplified as an internal control.
2.6. Immunofluorescence To further assess the myogenically-differentiated USCs with immunofluorescence, the cells cultured on chamber slides were fixed with 4.0% paraformaldehyde for 15–20 min at room temperature and permeabilized with 0.1% J Tissue Eng Regen Med (2014) DOI: 10.1002/term
W. Chen et al. Table 3. Primers for real-time PCR used in this study* Primers
Cell markers
Catalogue no.
Myf-5 MyoD Myosin Desmin GAPDH
Skeletal myogenic lineage Skeletal myogenic lineage Late myogenesis Myogenic lineage Housekeeping gene
Hs00224610 Hs00159528_m1 Hs00255652_m1 Hs01090875 NM_002046.3
*All primers were obtained from Applied Biosystems (Foster City, CA, USA).
Triton-100 in PBS for 3 min at room temperature and blocked. The cells were then incubated with primary antibodies (myf-5, myo D, myosin and desmin; Table 1), followed by an appropriate secondary antibody conjugated to fluorescein isothiocyanate. Finally, the slides were mounted in mounting medium (Vector Laboratories) before visualization under a fluorescent microscope. The sections stained with immunofluorescent specific myogenic markers were evaluated by two independent and blinded observers, using images captured by the microscope. The percentage of myogenically-induced USCs in total number of targeted cells was counted by semiquantitative assessment as the differentiation efficiency (%) in 10 random fields under × 200 magnification (Table 4).
before injection. Myogenically differentiated USCs or non-differentiated USCs (1 × 106 cells) were suspended in 100 μl PBS and injected into the tibialis anterior muscles of T/B cell-deficient SCID mice (n = 6). The injection procedure did not cause complications such as infection or bleeding. The animals were sacrificed after 4 weeks and the harvested tissue samples were immediately embedded in OCT compound for cryosectioning. Slides with 5 μm-thick sections were prepared for immunohistochemistry and fluorescent microscopy. Human specific nuclear antibody (NuMA; Abcam) was used to identify the implanted USCs prior to staining with other protein markers. Skeletal myogenic-specific antibodies (Myf5, myoD and myosin) were then used to characterize the expression of skeletal muscle markers in the injected cells in vivo. Myf5 and MyoD are required to determine skeletal myogenic lineages (Aurade et al., 1994; Rohwedel et al., 1994). Myosin is known as an element of skeletal muscle fibres that appears in late myogenesis (Gang et al., 2004). All experiments were conducted in accordance with institutional guidelines and the animal protocol was approved by the Institutional Animal Care and Use Committee of Wake Forest University Health Sciences.
2.9. Statistical analysis
2.7. Western blot To measure the myogenic proteins after induction of USCs, using western blotting, the cells were lysed with RIPA buffer (Sigma-Aldrich) supplemented with proteinase inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged at 1.2 × 104 g for 15 min at 4 °C. Protein (25–50 μg) was separated using a 10% sodium dodecyl sulphate– polyacrylamide (SDS–PAGE) gel and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline with 0.5% Tween-20 (TBST) containing 5% fat-free milk powder and then incubated with primary antibodies, followed by horseradish peroxidase (HP)conjugated secondary antibodies. Proteins were detected using the Super Signal West Femto chemiluminescence reagent (Pierce, Rockford, IL, USA) and the images were captured using a Fujifilm LAS-3000 Imager system.
2.8. In vivo studies HD differentiation medium was selected to induce USCs to differentiate into myogenic lineage cells for 4 weeks
Student’s t-tests and paired-sample t-tests were used for statistical analysis. Values are expressed as mean ± standard deviation (SD); p ≤ 0.05 was considered significant.
3. Results 3.1. USC proliferation and morphology Single clones of human USCs grew into a relatively homogeneous population in these experiments. The USCs proliferated rapidly, as seen by their doubling time (ca. 20–30 h; Table 2), with an average of 37 population doublings (range 32–57; i.e. 232–257 cells), leading to 1 × 1010–1 × 1017 cells within 5–6 weeks. The cells proliferated until p9–13, after which their doubling time started to increase (45 h) progressively with each passage until the cells stopped dividing. After induction, a characteristic feature of skeletal myogenic differentiated cells is a change in morphology from ’rice-grain’-like cells to a spindle shape similar to that of muscle cells, and the cells in HD medium changed significantly (Figure 1).
Table 4. Semi-qualitative analyses of differentiation efficiency in immunofluorescence analysis USC-P4
Myogenic differentiation efficiency of USCs (%)
Medium
KSFM/EFM
HD 4W
SK 4W
HD 2W
SK 2W
Myf5 MyoD Myosin Desmin
0 0 0 0
5.6 ± 1.9 4.9 ± 2.5 3.8 ± 2.7 4.1 ± 2.0
7.4 ± 2.6 6.8 ± 3.7 8.0 ± 5.1 9.6 ± 5.5
16.2 ± 4.9 20.2 ± 9.3 29.6 ± 8.8 30.5 ± 12.3
28.3 ± 5.1 43.3 ± 10.1 44.4 ± 14.1 40 ± 11.9
Copyright © 2014 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2014) DOI: 10.1002/term
Skeletal myogenic differentiation of hUSCs
Figure 1. Morphology of non-induced USCs and myogenic-induced USCs. Brightfield microscopy of non-induced USCs (p3) and induced USCs with HD medium for 2 weeks (HD2W) and 4 weeks (HD4W), SK medium for 2 weeks (SK2W) and 4 weeks (SK4W). Treatment with differentiation medium caused the cells to alter their morphology. Magnification = ×100
3.2. Cell surface marker expression USCs stained positive for the mesenchymal markers CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133 and STRO-1, and also stained positive for a pericyte marker, CD146, and an embryonic stem cell marker, homogenousSSEA-4. However, they stained negative for haematopoietic markers and endothelial surface markers, including CD14, CD34, CD45 and CD31 (Figure 2).
3.3. Myogenic differentiation of USCs in vitro
differentiation (Figure 3) compared to other induced differentiation groups. Immunofluorescent staining and western blotting using skeletal muscle-specific antibodies (myf5, myoD, myosin and desmin) revealed the expression of specific markers on induction with each of the two media compared to the non-induced USCs control (Figures 4, 5). The differentiated USCs in HD medium displayed myogenic markers more strongly than the cells induced in HD medium at 2 or 4 week periods in immunofluorescence analysis (Table 4). In addition, induced cells after 4 weeks of differentiation expressed skeletal musclespecific markers more strongly than cells induced by 2 weeks of differentiation, indicating the time-dependent effect of myogenic differentiation of USCs.
3.4. In vivo study The muscle tissue samples were collected 4 weeks after injection; as shown in Figure 6, more USCs expressed human nuclei and developed positive staining for the skeletal muscle-specific markers myf5, myoD and myosin in USCs differentiated by HD medium than non-induced USCs. Although less than induced USCs, non-induced USCs still stained positive for myogenic markers at the implanted sites and the USCs mainly appeared in the skeletal muscle interstitium 1 month after injection.
4. Discussion
To confirm the induction of myogenic transcripts, realtime PCR analysis was carried out using skeletal musclespecific primers. There was specific amplification in USC samples treated with HD medium after 4 weeks of
Mesenchyme stem cells are used as a cell source to repair defective or diseased tissues in two ways. First, by secreting paracrine factors, they can promote angiogenesis,
Figure 2. FCM analysis of USCs markers. USCs (p3) were analysed with MSC markers (CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133 and STRO-1), haematopoietic lineage markers (CD14, CD34, CD45), a pericyte marker (CD146), an embryonic stem cell marker (SSEA-4) and an endothelial-related lineage marker (CD31)
Figure 3. Real-time PCR analysis of skeletal myogenic differentiated USCs. USCs (p3) were differentiated using two differentiation media for 2 weeks (HD2W, SK2W) and 4 weeks (HD4W, SK4W) and analysed for expression of skeletal muscle-specific transcripts (myf5, myoD, myosin and desmin), using real-time PCR. Specific amplification was observed in induced samples in HD medium 4 weeks after induction. USCs (p4) and human skeletal muscle cells (SKMCs) were used as control and GAPDH as the housekeeping gene
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J Tissue Eng Regen Med (2014) DOI: 10.1002/term
W. Chen et al.
Figure 4. Immunofluorescent staining for skeletal muscle-specific markers. Skeletal muscle-specific markers in USCs following differentiation in vitro. Non-induced USCs and USCs induced with HD medium for 2 weeks (HD2W) and 4 weeks (HD4W), SK medium for 2 weeks (SK2W) and 4 weeks (SK4W), were stained with skeletal myogenic markers (Myf5, MyoD, myosin and desmin; red areas); nuclei were stained with DAPI (blue areas); non-induced USCs were used as control; scale bar = 50 μm
Figure 5. Western blot for skeletal muscle-specific markers. After induction with HD medium for 2 weeks (HD2W) and 4 weeks (HD4W), SK medium for 2 weeks (SK2W)and 4 weeks (SK4W), the differentiated USCs were analysed by western blot for skeletal muscle-specific markers (Myf5, MyoD, myosin and desmin), USCs (p4) were used as negative control and human skeletal muscle cells (SKMCs) were used as positive control
decrease fibrosis and recruit stem cells from the host tissues, to complete the repair, replacement and regeneration processes after implantation. In addition, surrounding normal cells and tissue-based signals from the host environment might guide the undifferentiated stem cells to give rise to the specific target cells required for tissue regeneration Copyright © 2014 John Wiley & Sons, Ltd.
(Baraniak and McDevitt, 2010; Furuta et al., 2007). Second, by trans-differentiation, stem cells are induced to differentiate into the target cells or tissue-like cells in vitro, and then implanted into defective sites for tissue repair and replacement where normal cells and tissues are not available (Chancellor et al., 2000). J Tissue Eng Regen Med (2014) DOI: 10.1002/term
Skeletal myogenic differentiation of hUSCs
Figure 6. Skeletal muscle differentiation of USCs in vivo. Immunofluorescent staining of sections harvested 1 month after injection into nude mice. USCs were identified using the human nuclear antibody (NuMA; red) and myogenic antibodies (FITC; green); the sections were also counterstained with DAPI to visualize all cells; scale bar = 50 μm
At present, few specific therapeutic options are available for the treatment of volumetric muscle loss (Sicari et al., 2012). More evidences demonstrate that several stem cells as a potential cell population have contributed to skeletal muscle regeneration (Sirabella et al., 2013; Tedesco et al., 2010), although their actual role in the regeneration process is still not clear. Our previous studies (Bodin et al., 2010; Zhang et al., 2008) demonstrated that USCs possess the stem cell property of plasticity and can differentiate to mesodermal lineages (osteocytes, chondrocytes, adipocytes and smooth muscle and endodermal cells) (Wu et al., 2011; Zhang et al., 2008). Therefore, it is very likely that USCs can be induced to differentiate into skeletal muscle lineage cells in myogenic differentiation medium. Our strategy in this study was to examine whether USCs could be guided to give rise to myogenic differentiation in vitro, and then maintain myogenic phenotypes in vivo, which can be potentially used to repair muscle deficiencies. We selected two from four different types of myogenic differentiation media in this study to induce the USCs to differentiate into skeletal muscle-like cells. The HD medium appeared to facilitate myogenic differentiation of USCs most efficiently after 4 weeks of differentiation in vitro, with a concomitant increase in the number of cells expressing myogenic gene and protein markers, compared to SK medium immunofluorescence analysis (Figures 3–5, Table 4). Although myotubes were still not as notable as in human skeletal cells in vitro, the gene and protein markers suggest that USCs can potentially differentiate into skeletal myoblasts. Interestingly, we found that a few non-preconditioned USCs (10–20%) also expressed myogenic markers at the implanted sites, indicating that the native muscle environment might play an important role in inducing the implanted USCs to differentiate into the skeletal myogenic lineage. Copyright © 2014 John Wiley & Sons, Ltd.
The receipt site for implanted cells is critical to determine the fate (i.e. survival and differentiation) of stem cells in vivo. In this study, our objective was to determine whether myogenically differentiated USCs can survive and retain myogenic phenotypes. To provide proof of concept, we injected the cells into the skeletal muscles of the legs of nude mice. Although not the same site as an injured skeletal muscle model for USCs to differentiate, we chose this site to simplify cell injection and monitoring the fate of injected cells. In future experiments, it will be necessary to evaluate the role of myogenic differentiation or the paracrine effect of USCs on skeletal muscle regeneration and tissue repair by implanting differentiated or nondifferentiated stem cells into muscle injury models (Stratos et al., 2011). The use of USCs as a cell source for cell therapy possesses several potential advantages compared to use of other MSCs, including the following: (a) USCs can be easily harvested by a non-invasive method and grown in culture; they do not require enzyme digestion or culture on a layer of feeder cells to support cell growth (Zhang et al., 2008); (b) USCs possess higher plasticity and more efficiency when induced to differentiate into other mesoderm cell types; (c) USCs can secrete angiogenic factors to promote revascularization; (d) USCs possess telomerase activity, allowing them to generate more cells and provide a longer life span for tissue regeneration; (e) USCs start as one single-cell colony in the initial culture, which creates more homogeneous cells after differentiation; and (f) as autologous cells, USCs do not raise ethical issues or cause immune reactions to engineered implants. Therefore, obtaining and using stem cells from urine could be an attractive alternative to stem cell-based therapy in tissue engineering (Bharadwaj et al., 2013). J Tissue Eng Regen Med (2014) DOI: 10.1002/term
W. Chen et al.
5. Conclusions USCs for use as pericytes/mesenchymal stem cells can be obtained easily by a non-invasive approach. The cells are able to expand extensively and can be induced to differentiate into a skeletal myogenic lineage. The myogenically differentiated USCs expressed Sk-MC gene and protein markers in vitro and in vivo. Our results demonstrated that HD medium can induce USCs to differentiate into skeletal lineage cells. The use of autologous USCs may serve as a more convenient and low-cost cell source, comparable in quality to other
types of adult stem cells, and could generate a large number of cells in vitro for potential use in cell injection therapy in skeletal muscle regeneration. For clinical applications, however, the functional capacity of these cells needs to be studied further.
Conflict of interest The authors have declared that there is no conflict of interest.
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using stem cells for stress urinary incontinence. Neurourol Urodyn 26: 966–971. Gang EJ, Jeong JA, Hong SH et al. 2004; Skeletal myogenic differentiation of mesenchymal stem cells isolated from human umbilical cord blood. Stem Cells 22: 617–624. Goudenege S, Pisani DF, Wdziekonski B et al. 2009; Enhancement of myogenic and muscle repair capacities of human adiposederived stem cells with forced expression of MyoD. Mol Ther 17: 1064–1072. Grabowska I, Brzoska E, Gawrysiak A et al. 2012; Restricted myogenic potential of mesenchymal stromal cells isolated from umbilical cord. Cell Transpl 21: 1711–1726. Grogan BF, Hsu JR, Skeletal Trauma Research Consortium. 2011; Volumetric muscle loss. J Am Acad Orthop Surg 19 (suppl 1): S35–S37. Huard J, Li Y, Fu FH. 2002; Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 84A: 822–832. Lang R, Liu G, Shi Y et al. 2013; Self-renewal and differentiation capacity of urinederived stem cells after urine preservation for 24 hours. PLoS One 8: e53980. Liadaki K, Casar JC, Wessen M et al. 2012; β4 integrin marks interstitial myogenic progenitor cells in adult murine skeletal muscle. J Histochem Cytochem 60: 31–44. Liu G, Pareta RA et al. 2013; Skeletal myogenic differentiation of urine-derived stem cells and angiogenesis using microbeads loaded with growth factors. Biomaterials 34: 1311–1326. Ma X, Zhang S, Zhou J et al. 2012; Clone-derived human AF-amniotic fluid stem cells are capable of skeletal myogenic differentiation in vitro and in vivo. J Tissue Eng Regen Med 6: 598–613. Merritt EK, Cannon MV, Hammers DW et al. 2010; Repair of traumatic skeletal muscle injury with bone-marrowderived mesenchymal stem cells seeded on extracellular matrix. Tissue Eng A 16: 2871–2881. Mitchell KJ, Pannerec A, Cadot B et al. 2010; Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development. Nat Cell Biol 12: 257–266. Mizuno H. 2010; The potential for treatment of skeletal muscle disorders with adipose-
derived stem cells. Curr Stem Cell Res Ther 5: 133–136. Moyer AL, Wagner KR. 2011; Regeneration versus fibrosis in skeletal muscle. Curr Opin Rheumatol 23: 568–573. Relaix F, Zammit PS. 2012; Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage. Development 139: 2845–2856. Rohwedel J, Maltsev V, Bober E et al. 1994; Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev Biol 164: 87–101. Sicari BM, Agrawal V, Siu BF et al. 2012; A murine model of volumetric muscle loss and a regenerative medicine approach for tissue replacement. Tissue Eng A 18: 1941–1948. Sirabella D, De Angelis L, Berghella L. 2013; Sources for skeletal muscle repair: from satellite cells to reprogramming. J Cachex Sarcopen Muscle 4: 125–136. Stern MM, Myers RL, Hammam N et al. 2009; The influence of extracellular matrix derived from skeletal muscle tissue on the proliferation and differentiation of myogenic progenitor cells ex vivo. Biomaterials 30: 2393–2399. Stratos I, Madry H, Rotter R et al. 2011; Fibroblast growth factor-2-overexpressing myoblasts encapsulated in alginate spheres increase proliferation, reduce apoptosis, induce adipogenesis, and enhance regeneration following skeletal muscle injury in rats. Tissue Eng A 17: 2867–2877. Tedesco FS, Dellavalle A, Diaz-Manera J et al. 2010; Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J Clin Invest 120: 11–19. Wu S, Liu Y, Bharadwaj S et al. 2011; Human urine-derived stem cells seeded in a modified 3D porous small intestinal submucosa scaffold for urethral tissue engineering. Biomaterials 32: 1317–1326. Zhang Y, McNeill E, Tian H et al. 2008; Urine derived cells are a potential source for urological tissue reconstruction. J Urol 180: 2226–2233. Zuk PA, Zhu M, Ashjian P et al. 2002; Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13: 4279–4295.
J Tissue Eng Regen Med (2014) DOI: 10.1002/term
ARTICLE
Skeletal myogenic differentiation of human urinederived cells as a potential source for skeletal muscle regeneration Wei Chen1,2, Minkai Xie1,3,4, Bin Yang1,5, Shantaram Bharadwaj1, Lujie Song1,3,4, Guihua Liu1, Shanhong Yi2, Gang Ye2, Anthony Atala1 and Yuanyuan Zhang1* 1
Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA Department of Urology, Xinqiao Hospital, Third Military Medical University, Chongqing, People’s Republic of China 3 Department of Urology, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai, People’s Republic of China 4 Shanghai Oriental Institute for Urologic Reconstruction, Shanghai, People’s Republic of China 5 Department of Urology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, People’s Republic of China 2
Abstract Stem cells are regarded as possible cell therapy candidates for skeletal muscle regeneration. However, invasive harvesting of those cells can cause potential harvest-site morbidity. The goal of this study was to assess whether human urine-derived stem cells (USCs), obtained through non-invasive procedures, can differentiate into skeletal muscle linage cells (Sk-MCs) and potentially be used for skeletal muscle regeneration. In this study, USCs were harvested from six healthy individuals aged 25–55. Expression profiles of cell-surface markers were assessed by flow cytometry. To optimize the myogenic differentiation medium, we selected two from four different types of myogenic differentiation media to induce the USCs. Differentiated USCs were identified with myogenic markers by gene and protein expression. USCs were implanted into the tibialis anterior muscles of nude mice for 1 month. The results showed that USCs displayed surface markers with positive staining for CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133, CD146, SSEA-4 and STRO-1, and negative staining for CD14, CD31, CD34 and CD45. After myogenic differentiation, a change in morphology was observed from ‘rice-grain’-like cells to spindle-shaped cells. The USCs expressed specific Sk-MC transcripts and protein markers (myf5, myoD, myosin, and desmin) after being induced with different myogenic culture media. Implanted cells expressed Sk-MC markers stably in vivo. Our findings suggest that USCs are able to differentiate into the Sk-MC lineage in vitro and after being implanted in vivo. Thus, they might be a potential source for cell injection therapy in the use of skeletal muscle regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Received 10 June 2013; Revised 11 March 2014; Accepted 20 April 2014
Keywords urine-derived stem cells; myogenic regeneration; tissue engineering; stem cells; differentiation; cell therapy
1. Introduction Skeletal muscle is one of the major tissues, comprising 40–50% of the human body by weight (Huard et al., 2002). Skeletal muscle has a robust capacity to repair itself, based on the satellite cells that are localized between the muscle fibre sarcolemma and the surrounding *Correspondence to: Yuanyuan Zhang, Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, 391 Technology Way, Winston-Salem, NC 27157, USA. E-mail: [email protected] Copyright © 2014 John Wiley & Sons, Ltd.
basal lamina (Cossu and Biressi, 2005; Relaix and Zammit, 2012). However, when there is loss of volumetric muscle, as in congenital muscle dystrophy or severe muscle trauma, scar tissue will accumulate because of a lack of satellite cells, leading to a myogenic dysfunction (Grogan et al., 2011; Moyer and Wagner, 2011). Autologous adult stem cell injection therapy has provided a promising alternative for skeletal muscle regeneration. Progenitor cells or stem cells obtained from skeletal muscle (Liadaki et al., 2012; Mitchell et al., 2010), bone marrow (de la Garza-Rodea et al., 2011; Merritt
W. Chen et al.
et al., 2010), umbilical cord (Grabowska et al., 2012), adipose tissues (Goudenege et al., 2009; Mizuno, 2010), amniotic fluid (Ma et al., 2012) and, more recently, induced pluripotent cells (iPS) (Darabi et al., 2012) are regarded as possible candidates for use in this therapy. However, harvesting these types of cells is invasive and may cause complications and donor-site morbidity. An autologous stem cell source that can be obtained using non-invasive techniques would be highly desirable. Recently, we demonstrated that a subpopulation of urine-derived cells, termed urine-derived stem cells (USCs), possess stem cell capabilities, such as selfrenewal, paracrine effect, immunomodulatory properties and multipotential differentiation. These cells can differentiate into mesodermal cell lineages, such as osteocytes, chondrocytes, adipocytes, endothelial cells and myocytes, including smooth muscle cell differentiation and endodermal lineages (e.g. urothelial cells) (Bharadwaj et al., 2013; Lang et al., 2013; Liu et al., 2013; Wu et al., 2011; Zhang et al., 2008). These cells maintain higher telomerase activity and possess long telomeres; further, they retain a normal karyotype in culture medium containing epidermal growth factors, even after several passages. Importantly, these cells do not form teratomas in vivo. USCs express cell surface markers associated with pericytes and mesenchymal stem cells (Bharadwaj et al., 2011; Bodin et al., 2010; Zhang et al., 2008). These cells can be isolated from regular voided urine (Zhang et al., 2008) from each individual via a noninvasive, simple and low-cost approach. The USCs isolated from one single urine specimen can generate 50–100 million cells at early passage (P4) in 3–4 weeks, sufficient numbers to use for cell therapy (Zhang et al., 2008). Thus, the goal of this study was to assess whether USCs, obtained though simple, non-invasive procedures, can differentiate into skeletal muscle cells (Sk-MCs) and potentially be used to treat skeletal muscle regeneration.
70–80% confluence, the cells were trypsinized and transferred into six-well dishes (P1). When the cultures reached 70–80% confluence once more, the cells were transferred to a 100 mm culture dish for expansion. Cell clones (P3) were selected for testing the mesenchymal/ pericyte surface markers with flow cytometry (FCM). The USCs clones were analysed with MSC markers (CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133 and STRO-1), haematopoietic lineage markers (CD14,CD34,CD45), a pericyte marker (CD146), an embryonic stem cell marker (SSEA-4) and an endothelialrelated lineage marker (CD31) (Table 1). Human Sk-MCs as control were isolated from healthy gracilis muscles discarded from surgery, as previously described (Liu et al., 2013). Briefly, the muscle samples were minced into small pieces, then incubated in collagenase II (0.1% w/v)–dispase (4 mg/ml) solution prepared in Dulbecco’s minimum essential medium (DMEM) for 1 h at 37 °C with constant shaking (60 rpm). The liberated cells were collected (400 × g) and suspended in DMEM containing 10% horse serum and plated into a six-well tissue culture dish. After 5 days in culture, the medium was changed to SkGM2 (Lonza Biologics, Portsmouth, NH, USA) containing 10% FBS at 37 °C in a 5% CO2 cell incubator.
2.2. Proliferation assay USCs were cultured at a density of 5000 cells/cm2, with medium changes every 2 days. Cells were trypsinized and manually counted in a haemocytometer when the cell density reached 70–80% confluence (generally 3–5 days). The population doubling (PD) and doubling times (DT) were calculated using the following formulae (Table 2):
Table 1. Antibodies used in this study and their dilutions
2. Materials and methods 2.1. Cell culture The protocol for obtaining human urine samples for this study was approved by the Wake Forest University Institutional Review Board. USCs were isolated and characterized from 15 individual urine samples collected from six healthy individuals aged 25–55 years, as previously described (Bodin et al., 2010; Zhang et al., 2008). Briefly, voided urine samples were centrifuged at 500 × g for 5 min and the supernatant was removed. The cell pellet was gently resuspended in medium comprising of keratinocyte serum-free medium (KSFM) and embryonic fibroblast medium (EFM), mixed at a ratio of 1:1, and 5% fetal bovine serum (FBS) and plated in 24-well plates at 1 × 104 cells/well. Individual clones appeared 4–7 days after implantation. Each clone was plated into a single well of a 24-well dish, and when the well reached Copyright © 2014 John Wiley & Sons, Ltd.
Primary antibody
Source/cat. no.
Antibody dilution
CD14-APC CD24-FITC CD29-PE CD31-FITC CD34-FITC CD44-PE CD45-APC CD73-PE CD90-APC CD105-PE CD117-PE CD133-PE CD146-PE STRO-1-FITC SSEA-4-PE MsIgG1-PE MsIgG1-FITC MsIgG1-APC Myf5 (C-20) MyoD (C-20) Myosin (G-4) Desmin (RD301) NuMA
R&D Systems/FAB3832A BD Biosciences/BD 560992 BD Biosciences/BD 556049 BD Biosciences/BD 560984 BD Biosciences/BD 348053 BD Biosciences/BD 550989 BD Biosciences/BD 340943 BD Biosciences/BD550257 BD Biosciences/BD 559869 BD Biosciences/BD 560839 BD Biosciences/BD 340867 Miltenyi Biotec/MACS 130080801 BD Biosciences/BD 550315 Biolegend /340105 BD Biosciences/BD 560128 BD Biosciences/BD 559320 BD Biosciences/BD 555909 BD Biosciences/BD 555751 Santa Cruz/sc302 Santa Cruz/sc304 Santa Cruz/sc6956 Santa Cruz/sc23879 Millipore/mab1281
1:20 1:10 1:10 1:10 1:10 1:10 1:40 1:40 1:10 1:40 1:10 1:20 1:10 1:40 1:10 1:10 1:10 1:10 1:100 1:100 1:100 1:100 1:100
J Tissue Eng Regen Med (2014) DOI: 10.1002/term
Skeletal myogenic differentiation of hUSCs Table 2. Proliferative ability of USC clones USC clone A2 D3 B3
Average doubling time (DT)
Total population doublings (PD)
Starting cell number
Ending cell number
24.9 ± 9.4 29.9 ± 10.3 20.7 ± 4.1
56.7 48.0 32.2
1 1 1
1.17 × 10 14 2.79 × 10 9 4.89 × 10
PD = ln(Nf/Ni)/ln(2) and DT = Ct/PD, where Nf is the final number of cells, Ni is the initial number of cells and Ct is the culture time.
2.3. FACS analysis USCs (P3) were trypsinized and washed in phosphatebuffered saline (PBS) containing 0.5% bovine serum albumin (BSA). One million cells in 100 ml PBS containing 3% BSA were incubated with fluorochome-conjugated antibodies (Table 1) for 30 min at 4 °C. The cells were washed again, resuspended in 100 ml PBS containing 0.5% BSA (three times) and passed through a 70 mm cell filter before flow analysis using a FACS Calibur machine (BD Biosciences, Franklin Lakes, NJ, USA).
2.4. Optimization of myogenic culture medium Several media and supplements have been suggested for stem cell differentiation to a skeletal muscle lineage (De Coppi et al., 2007; Zuk et al., 2002). In the present study, USCs were induced to differentiate into Sk-MCs using the four different media listed below: 1. HD differentiation medium: DMEM (2 mM L-glutamine), 10% FBS, 5% horse serum, 1% penicillin–streptomycin, 50 μM hydrocortisone, 0.1 μM dexamethasone (Gibco/BRL). USCs (p3) were plated at a density of 1 × 104 cells/cm2 ™ on plates coated with Matrigel (1:1 diluted with medium; Collaborative Biomedical Products) and exposed to HD medium. The media were changed every 3 days up to 2 weeks (HD2W) and 4 weeks (HD4W) (Zuk et al., 2002).
2. SK differentiation medium: Sk-MC growth medium (SkGM-2, Lonza), followed by differentiation medium [DMEM:F-12 (1:1), 2% horse serum, 1% penicillin– streptomycin (Gibco/BRL). USCs (P3) were seeded at a density of 1 × 104 cells/cm2 on plastic plates precoated with Matrigel (1:1 diluted with medium) and exposed to SkGM-2 for 1 week, followed by differentiation medium. The differentiation medium was changed every 3 days up to 2 weeks (SK2W) and 4 weeks (SK4W) (Stern et al., 2009). 3. 1% CEE differentiation medium: DMEM (2 mM L-glutamine), 10% horse serum, 1% penicillin–streptomycin, 1.0% chick embryo extract (CEE; Gibco/BRL), 3 μM 5′Aza 2′deoxy cytidine (5-azaC; Sigma-Aldrich). USCs (P3) were seeded at a density of 1 × 104 cells/cm2 on plates precoated with Matrigel (1:1 diluted with medium) in Copyright © 2014 John Wiley & Sons, Ltd.
Total time for expansion (days)
Number of passages
55 51 30
13 11 9
17
1% CEE medium without 5-azaC; 12 h after seeding, 5-azaC was added to the culture medium for 24 h of incubation, then incubation continued in complete medium lacking 5-azaC, with medium changes every 3 days up to 28 days (De Coppi et al., 2007). 4. Conditioned differentiation medium: spent media from primary cultured human Sk-MCs. Conditioned medium was prepared from differentiated human skeletal muscle cells cultured in DMEM:F-12 (1:1) containing 2% horse serum and 1% penicillin–streptomycin at a confluence of 60–80%. The spent medium (15–18 h) was filtered (0.2 μM ) and used immediately or stored at –80 °C until further use. The USCs were plated at 1 × 104 cells/cm 2 on Matrigel (1:1 diluted with medium) with conditioned medium and then the medium was changed every 3–4 days up to 4 weeks. After optimization of the four induced media, our results showed that HD and SK differentiation media could induce USCs in myogenic lineage gene and protein expression at higher levels than the other two media (data not shown). We therefore focused on both HD and SK differentiation media for further analysis.
2.5. RNA analysis To determine myogenic differentiation of USCs with realtime PCR, the mRNA was extracted with an RNA isolation kit (5 PRIMEs, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Total RNA (5 μg) was converted to cDNA in a reaction containing random primers, nucleotides and reverse transcriptase enzymes, using a high-capacity cDNA reverse-transcription kit (Applied Biosystems, Foster City, CA, USA). One-tenth of the cDNA was then used for real-time analysis, along with Taqman Universal PCR Master Mix and Taqman gene expression probes, according to the manufacturer’s instructions, using a 7300 Real-time PCR system (Applied Biosystems). The primer pairs used in this study are listed in Table 3. To normalize mRNA levels, the GAPDH housekeeping gene was amplified as an internal control.
2.6. Immunofluorescence To further assess the myogenically-differentiated USCs with immunofluorescence, the cells cultured on chamber slides were fixed with 4.0% paraformaldehyde for 15–20 min at room temperature and permeabilized with 0.1% J Tissue Eng Regen Med (2014) DOI: 10.1002/term
W. Chen et al. Table 3. Primers for real-time PCR used in this study* Primers
Cell markers
Catalogue no.
Myf-5 MyoD Myosin Desmin GAPDH
Skeletal myogenic lineage Skeletal myogenic lineage Late myogenesis Myogenic lineage Housekeeping gene
Hs00224610 Hs00159528_m1 Hs00255652_m1 Hs01090875 NM_002046.3
*All primers were obtained from Applied Biosystems (Foster City, CA, USA).
Triton-100 in PBS for 3 min at room temperature and blocked. The cells were then incubated with primary antibodies (myf-5, myo D, myosin and desmin; Table 1), followed by an appropriate secondary antibody conjugated to fluorescein isothiocyanate. Finally, the slides were mounted in mounting medium (Vector Laboratories) before visualization under a fluorescent microscope. The sections stained with immunofluorescent specific myogenic markers were evaluated by two independent and blinded observers, using images captured by the microscope. The percentage of myogenically-induced USCs in total number of targeted cells was counted by semiquantitative assessment as the differentiation efficiency (%) in 10 random fields under × 200 magnification (Table 4).
before injection. Myogenically differentiated USCs or non-differentiated USCs (1 × 106 cells) were suspended in 100 μl PBS and injected into the tibialis anterior muscles of T/B cell-deficient SCID mice (n = 6). The injection procedure did not cause complications such as infection or bleeding. The animals were sacrificed after 4 weeks and the harvested tissue samples were immediately embedded in OCT compound for cryosectioning. Slides with 5 μm-thick sections were prepared for immunohistochemistry and fluorescent microscopy. Human specific nuclear antibody (NuMA; Abcam) was used to identify the implanted USCs prior to staining with other protein markers. Skeletal myogenic-specific antibodies (Myf5, myoD and myosin) were then used to characterize the expression of skeletal muscle markers in the injected cells in vivo. Myf5 and MyoD are required to determine skeletal myogenic lineages (Aurade et al., 1994; Rohwedel et al., 1994). Myosin is known as an element of skeletal muscle fibres that appears in late myogenesis (Gang et al., 2004). All experiments were conducted in accordance with institutional guidelines and the animal protocol was approved by the Institutional Animal Care and Use Committee of Wake Forest University Health Sciences.
2.9. Statistical analysis
2.7. Western blot To measure the myogenic proteins after induction of USCs, using western blotting, the cells were lysed with RIPA buffer (Sigma-Aldrich) supplemented with proteinase inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged at 1.2 × 104 g for 15 min at 4 °C. Protein (25–50 μg) was separated using a 10% sodium dodecyl sulphate– polyacrylamide (SDS–PAGE) gel and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline with 0.5% Tween-20 (TBST) containing 5% fat-free milk powder and then incubated with primary antibodies, followed by horseradish peroxidase (HP)conjugated secondary antibodies. Proteins were detected using the Super Signal West Femto chemiluminescence reagent (Pierce, Rockford, IL, USA) and the images were captured using a Fujifilm LAS-3000 Imager system.
2.8. In vivo studies HD differentiation medium was selected to induce USCs to differentiate into myogenic lineage cells for 4 weeks
Student’s t-tests and paired-sample t-tests were used for statistical analysis. Values are expressed as mean ± standard deviation (SD); p ≤ 0.05 was considered significant.
3. Results 3.1. USC proliferation and morphology Single clones of human USCs grew into a relatively homogeneous population in these experiments. The USCs proliferated rapidly, as seen by their doubling time (ca. 20–30 h; Table 2), with an average of 37 population doublings (range 32–57; i.e. 232–257 cells), leading to 1 × 1010–1 × 1017 cells within 5–6 weeks. The cells proliferated until p9–13, after which their doubling time started to increase (45 h) progressively with each passage until the cells stopped dividing. After induction, a characteristic feature of skeletal myogenic differentiated cells is a change in morphology from ’rice-grain’-like cells to a spindle shape similar to that of muscle cells, and the cells in HD medium changed significantly (Figure 1).
Table 4. Semi-qualitative analyses of differentiation efficiency in immunofluorescence analysis USC-P4
Myogenic differentiation efficiency of USCs (%)
Medium
KSFM/EFM
HD 4W
SK 4W
HD 2W
SK 2W
Myf5 MyoD Myosin Desmin
0 0 0 0
5.6 ± 1.9 4.9 ± 2.5 3.8 ± 2.7 4.1 ± 2.0
7.4 ± 2.6 6.8 ± 3.7 8.0 ± 5.1 9.6 ± 5.5
16.2 ± 4.9 20.2 ± 9.3 29.6 ± 8.8 30.5 ± 12.3
28.3 ± 5.1 43.3 ± 10.1 44.4 ± 14.1 40 ± 11.9
Copyright © 2014 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2014) DOI: 10.1002/term
Skeletal myogenic differentiation of hUSCs
Figure 1. Morphology of non-induced USCs and myogenic-induced USCs. Brightfield microscopy of non-induced USCs (p3) and induced USCs with HD medium for 2 weeks (HD2W) and 4 weeks (HD4W), SK medium for 2 weeks (SK2W) and 4 weeks (SK4W). Treatment with differentiation medium caused the cells to alter their morphology. Magnification = ×100
3.2. Cell surface marker expression USCs stained positive for the mesenchymal markers CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133 and STRO-1, and also stained positive for a pericyte marker, CD146, and an embryonic stem cell marker, homogenousSSEA-4. However, they stained negative for haematopoietic markers and endothelial surface markers, including CD14, CD34, CD45 and CD31 (Figure 2).
3.3. Myogenic differentiation of USCs in vitro
differentiation (Figure 3) compared to other induced differentiation groups. Immunofluorescent staining and western blotting using skeletal muscle-specific antibodies (myf5, myoD, myosin and desmin) revealed the expression of specific markers on induction with each of the two media compared to the non-induced USCs control (Figures 4, 5). The differentiated USCs in HD medium displayed myogenic markers more strongly than the cells induced in HD medium at 2 or 4 week periods in immunofluorescence analysis (Table 4). In addition, induced cells after 4 weeks of differentiation expressed skeletal musclespecific markers more strongly than cells induced by 2 weeks of differentiation, indicating the time-dependent effect of myogenic differentiation of USCs.
3.4. In vivo study The muscle tissue samples were collected 4 weeks after injection; as shown in Figure 6, more USCs expressed human nuclei and developed positive staining for the skeletal muscle-specific markers myf5, myoD and myosin in USCs differentiated by HD medium than non-induced USCs. Although less than induced USCs, non-induced USCs still stained positive for myogenic markers at the implanted sites and the USCs mainly appeared in the skeletal muscle interstitium 1 month after injection.
4. Discussion
To confirm the induction of myogenic transcripts, realtime PCR analysis was carried out using skeletal musclespecific primers. There was specific amplification in USC samples treated with HD medium after 4 weeks of
Mesenchyme stem cells are used as a cell source to repair defective or diseased tissues in two ways. First, by secreting paracrine factors, they can promote angiogenesis,
Figure 2. FCM analysis of USCs markers. USCs (p3) were analysed with MSC markers (CD24, CD29, CD44, CD73, CD90, CD105, CD117, CD133 and STRO-1), haematopoietic lineage markers (CD14, CD34, CD45), a pericyte marker (CD146), an embryonic stem cell marker (SSEA-4) and an endothelial-related lineage marker (CD31)
Figure 3. Real-time PCR analysis of skeletal myogenic differentiated USCs. USCs (p3) were differentiated using two differentiation media for 2 weeks (HD2W, SK2W) and 4 weeks (HD4W, SK4W) and analysed for expression of skeletal muscle-specific transcripts (myf5, myoD, myosin and desmin), using real-time PCR. Specific amplification was observed in induced samples in HD medium 4 weeks after induction. USCs (p4) and human skeletal muscle cells (SKMCs) were used as control and GAPDH as the housekeeping gene
Copyright © 2014 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med (2014) DOI: 10.1002/term
W. Chen et al.
Figure 4. Immunofluorescent staining for skeletal muscle-specific markers. Skeletal muscle-specific markers in USCs following differentiation in vitro. Non-induced USCs and USCs induced with HD medium for 2 weeks (HD2W) and 4 weeks (HD4W), SK medium for 2 weeks (SK2W) and 4 weeks (SK4W), were stained with skeletal myogenic markers (Myf5, MyoD, myosin and desmin; red areas); nuclei were stained with DAPI (blue areas); non-induced USCs were used as control; scale bar = 50 μm
Figure 5. Western blot for skeletal muscle-specific markers. After induction with HD medium for 2 weeks (HD2W) and 4 weeks (HD4W), SK medium for 2 weeks (SK2W)and 4 weeks (SK4W), the differentiated USCs were analysed by western blot for skeletal muscle-specific markers (Myf5, MyoD, myosin and desmin), USCs (p4) were used as negative control and human skeletal muscle cells (SKMCs) were used as positive control
decrease fibrosis and recruit stem cells from the host tissues, to complete the repair, replacement and regeneration processes after implantation. In addition, surrounding normal cells and tissue-based signals from the host environment might guide the undifferentiated stem cells to give rise to the specific target cells required for tissue regeneration Copyright © 2014 John Wiley & Sons, Ltd.
(Baraniak and McDevitt, 2010; Furuta et al., 2007). Second, by trans-differentiation, stem cells are induced to differentiate into the target cells or tissue-like cells in vitro, and then implanted into defective sites for tissue repair and replacement where normal cells and tissues are not available (Chancellor et al., 2000). J Tissue Eng Regen Med (2014) DOI: 10.1002/term
Skeletal myogenic differentiation of hUSCs
Figure 6. Skeletal muscle differentiation of USCs in vivo. Immunofluorescent staining of sections harvested 1 month after injection into nude mice. USCs were identified using the human nuclear antibody (NuMA; red) and myogenic antibodies (FITC; green); the sections were also counterstained with DAPI to visualize all cells; scale bar = 50 μm
At present, few specific therapeutic options are available for the treatment of volumetric muscle loss (Sicari et al., 2012). More evidences demonstrate that several stem cells as a potential cell population have contributed to skeletal muscle regeneration (Sirabella et al., 2013; Tedesco et al., 2010), although their actual role in the regeneration process is still not clear. Our previous studies (Bodin et al., 2010; Zhang et al., 2008) demonstrated that USCs possess the stem cell property of plasticity and can differentiate to mesodermal lineages (osteocytes, chondrocytes, adipocytes and smooth muscle and endodermal cells) (Wu et al., 2011; Zhang et al., 2008). Therefore, it is very likely that USCs can be induced to differentiate into skeletal muscle lineage cells in myogenic differentiation medium. Our strategy in this study was to examine whether USCs could be guided to give rise to myogenic differentiation in vitro, and then maintain myogenic phenotypes in vivo, which can be potentially used to repair muscle deficiencies. We selected two from four different types of myogenic differentiation media in this study to induce the USCs to differentiate into skeletal muscle-like cells. The HD medium appeared to facilitate myogenic differentiation of USCs most efficiently after 4 weeks of differentiation in vitro, with a concomitant increase in the number of cells expressing myogenic gene and protein markers, compared to SK medium immunofluorescence analysis (Figures 3–5, Table 4). Although myotubes were still not as notable as in human skeletal cells in vitro, the gene and protein markers suggest that USCs can potentially differentiate into skeletal myoblasts. Interestingly, we found that a few non-preconditioned USCs (10–20%) also expressed myogenic markers at the implanted sites, indicating that the native muscle environment might play an important role in inducing the implanted USCs to differentiate into the skeletal myogenic lineage. Copyright © 2014 John Wiley & Sons, Ltd.
The receipt site for implanted cells is critical to determine the fate (i.e. survival and differentiation) of stem cells in vivo. In this study, our objective was to determine whether myogenically differentiated USCs can survive and retain myogenic phenotypes. To provide proof of concept, we injected the cells into the skeletal muscles of the legs of nude mice. Although not the same site as an injured skeletal muscle model for USCs to differentiate, we chose this site to simplify cell injection and monitoring the fate of injected cells. In future experiments, it will be necessary to evaluate the role of myogenic differentiation or the paracrine effect of USCs on skeletal muscle regeneration and tissue repair by implanting differentiated or nondifferentiated stem cells into muscle injury models (Stratos et al., 2011). The use of USCs as a cell source for cell therapy possesses several potential advantages compared to use of other MSCs, including the following: (a) USCs can be easily harvested by a non-invasive method and grown in culture; they do not require enzyme digestion or culture on a layer of feeder cells to support cell growth (Zhang et al., 2008); (b) USCs possess higher plasticity and more efficiency when induced to differentiate into other mesoderm cell types; (c) USCs can secrete angiogenic factors to promote revascularization; (d) USCs possess telomerase activity, allowing them to generate more cells and provide a longer life span for tissue regeneration; (e) USCs start as one single-cell colony in the initial culture, which creates more homogeneous cells after differentiation; and (f) as autologous cells, USCs do not raise ethical issues or cause immune reactions to engineered implants. Therefore, obtaining and using stem cells from urine could be an attractive alternative to stem cell-based therapy in tissue engineering (Bharadwaj et al., 2013). J Tissue Eng Regen Med (2014) DOI: 10.1002/term
W. Chen et al.
5. Conclusions USCs for use as pericytes/mesenchymal stem cells can be obtained easily by a non-invasive approach. The cells are able to expand extensively and can be induced to differentiate into a skeletal myogenic lineage. The myogenically differentiated USCs expressed Sk-MC gene and protein markers in vitro and in vivo. Our results demonstrated that HD medium can induce USCs to differentiate into skeletal lineage cells. The use of autologous USCs may serve as a more convenient and low-cost cell source, comparable in quality to other
types of adult stem cells, and could generate a large number of cells in vitro for potential use in cell injection therapy in skeletal muscle regeneration. For clinical applications, however, the functional capacity of these cells needs to be studied further.
Conflict of interest The authors have declared that there is no conflict of interest.
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J Tissue Eng Regen Med (2014) DOI: 10.1002/term
