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Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture
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Research Article
Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture Yan Wei a,b , Yuan Li b , Chao Chen a , Katharina Stoelzel a , Andreas M. Kaufmann c , Andreas E. Albers a,⁎ a
Department of Otolaryngology, Head and Neck Surgery Charité-Universitätsmedizin Berlin, Berlin, Germany Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou, China c Clinic for Gynecology CCM/CBF, Charité-Universitätsmedizin Berlin, Berlin, Germany b
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Human skeletal muscle contains an accessible adult stem-cell compartment in which
Received 17 September 2010
differentiated myofibers are maintained and replaced by a self-renewing stem cell pool.
Revised version received
Previously, studies using mouse models have established a critical role for resident stem cells in
14 January 2011
skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the
Accepted 15 January 2011
reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate
Available online 27 January 2011
for extended periods of time as floating clusters of rounded cells, termed “myospheres” or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional
Keywords:
properties of these cells were determined using reverse transcription-polymerase chain reaction
Muscular dystrophy
(RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are
Satellite cell
clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem
Myosphere
cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be
Induced differentiation
maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times,
Self-renewal
despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were
Adult stem cell
successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types. © 2011 Elsevier Inc. All rights reserved.
⁎ Corresponding author at: Hals-Nasen-Ohrenklinik, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200, Berlin. E-mail address: [email protected] (A.E. Albers). Abbreviations: MDPCs, myosphere-derived progenitor cells; ALDH1, aldehyde dehydrogenase-1; SCs, satellite cell; TFs, transcription factors; FACS, fluorescence-activated cell sorting; SMA, smooth muscle alpha-actin; iPSCs, induced pluripotent stem cells; MGM, myosphere-growing medium; MPM, muscle proliferation medium; MFM, muscle fusion media; RT-PCR, reverse transcription-polymerase chain reaction; CPD, cumulative population doubling; PDT, population doubling time 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.01.019
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Introduction Therapeutic approaches for skeletal muscle degeneration by cell transplantation have been impeded so far by low numbers of accessible cells, poor cellular survival rates, and lack of reproducible methods of expansion [1–4]. Ideal graft cells should be nonimmunogenic, easy to expand in vitro, and be able to engraft and differentiate into functional myotubes. Satellite cells (SCs) are quiescent cells that are located beneath the basal lamina of skeletal muscle fibers. They are recruited for muscle growth, maintenance, repair, and regeneration [5]. So far, difficulties in studying human skeletal muscle arose, not only because of practical difficulties in obtaining muscle biopsies to prepare cells in sufficient quantity, but also due to the lack of markers suitable to distinguish satellite cells from myonuclei and other cells present within the skeletal muscle. That is why most research so far relied on animal studies focusing on rodents, particularly mice. Moreover, directly isolated SCs are of low quantity and an expansion step is highly desirable. The spontaneous formation of floating spheres, by three dimensional aggregation of cells cultivated under ultra-low adhesion conditions, is a known prerogative of neural stem cells, endothelial stem cells, and cardiomyocytes [6–8]. These culture methods imitate the three-dimensional structure of tissue and involve as little manipulation as possible to maintain the cellular differentiation potential. In suspension culture, differentiated cells stop dividing and die whereas stem cells continue to proliferate in an apparently asymmetric way by giving rise to many secondary spheres with an exponential growth rate [9]. Recent research demonstrated that muscle stem cells could be isolated and expanded from mice by forming myospheres, which have the potential capability of selfrenewal and multi-lineage differentiation [10,11]. However, to our knowledge, no similar stem cell research involving an isolation and expansion step has been reported concerning human skeletal muscle. So far, only CD56 and Pax7 have been extensively used for identification of SCs on sections of human muscle [12,13]. More recently, Vauchez and colleagues demonstrated that aldehyde dehydrogenase (ALDH) activity was a novel marker for a population of human skeletal muscle progenitors [14]. Investigations confirmed that the SCs are a heterogeneous cell population and only a subset of SCs exhibited asymmetric cell division, longterm Bromodeoxyuridine (BrdU) retention, and appear to be the progenitors of satellite cells [15–19]. It was reported that Oct4, Sox2, and Nanog, which form a self-organized core of transcription factors (TFs), maintain pluripotency and self-renewal capacity of embryonic stem cells [20,21]. More recently, expression of these TFs in adult stem cells suggests, that these stem cells are farther upstream in the line of differentiation [22–24]. Nevertheless, until now, nothing has been reported about the relationship between these TFs and human muscle stem cells. In this study, we prospectively isolated and expanded cells by generating “myospheres” that consist of myosphere-derived progenitor cells (MDPCs) from human skeletal muscle samples. MDPCs maintain the capacity of self-renewal, proliferation and multipotentiality. Our studies also indicate that heterogeneity in the expression of molecular markers in MDPCs may reflect a functional diversity within these cell populations. This observation is further supported by differential expression of TFs. Considering their unproblematic isolation and their proliferative potential, MDPCs could have a significant impact on future clinical strategies to treat patients with muscular disease.
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Materials and methods Human skeletal muscle Skeletal muscle biopsies were obtained from the omohyoid muscle of 20 patients (Supplementary Table 1), who underwent surgery at the Department for Otolaryngology, Head and Neck surgery, Campus Benjamin Franklin, Charité, Universitätsmedizin, Berlin, Germany. Most patients underwent surgery for the resection of cervical lymphnodes as indicated by the presence of a head and neck carcinoma. During this surgery the omohyoid muscle is often cut for optimal surgical exposure. Biopsies were typically taken from one of the free ends. To exclude any infiltration of the muscle tissues, only those cases were selected, where the resection margins were free of cancer as stated by the pathologist. The patients were free of known muscular pathologies. The study was carried out with the approval of the institutional ethics committee (IRB #EA4/033/09). Informed consent was obtained from each patient whose tissues were collected for the study.
Isolation of adult human muscle stem cells Skeletal-muscle stem cells were isolated from the muscle biopsies (0.1–1 g) by a procedure similar to that previously described for human muscle [25]. Briefly, the skeletal muscle tissue was minced into 1–3 mm3 pieces and washed with phosphate buffered saline without Ca2+/Mg2+ (PBS) several times to remove fat and contaminating red blood cells. Muscle biopsies were then finely minced and digested for 60 to 120 min at 37 °C with a mixture of type-I and type-IV collagenase (100 μg/ml) and dispase (1.2 μg/ml; all from GIBCO, Invitrogen, Darmstadt, Germany). The digested tissue was mechanically dissociated and then passed through a 40 μm mesh to obtain a single cell suspension. The single cell suspensions were plated in Corning* Ultra-Low Attachment 6 well plates (Fisher Scientific, Loughborough, UK) containing a myosphere-growing medium (MGM: DMEM/F12, EGF 20 ng/ml, bFGF 20 ng/ml) and kept at humidified atmosphere of 5% CO2 in air.
Myosphere formation and expansion Fresh MGM was added to the cultures every second day (500 μl per well). After 5 days, the suspension was transferred into 50 ml conical tubes and the cell clusters were allowed to sediment for 30 min. Then half of the medium was replaced with new MGM and the cells were replated. After 7–10 days cells proliferated to form floating myospheres. Myospheres were picked with a 40 μm mesh from the media and transferred into a collagen type I (SigmaAldrich, Munich, Germany) coated culture flask in muscle proliferation media (MPM: DMEM/F12, 20% FCS, bFGF 5 ng/ml, 1% P/S). When the cultures approached 60% confluency the MDPCs were digested and passaged at a ratio of 1:2.
Growth rate To evaluate the growth rate of MDPCs, cells were seeded at 3000 cells/cm2 in 25 cm2 tissue culture flasks (Sarsted, Numbrecht, Germany) in MPM. Cells were detached using 0.25% trypsin/EDTA (Biochrom AG, Berlin, Germany) every 5–10 days and the cell
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number was determined. This procedure was repeated over a period of 20 weeks. The calculated population doublings were summarized to compute the cumulative population doubling (CPD) for each of the three cell samples. We calculated the population doubling time (PDT) as previously described [26] by calculating PDT = T/(log2(NF/N0)), where T = time in culture, N0 = initial number of cells, and NF = final number of cells.
Immunocytochemistry Adherent cells from passage 3, 5, 6, 8 and 10 were grown on lysine (0.1 mg/ml, Biochrom AG) coated glass coverslips respectively in MPM. Intact myospheres were put on a glass slide using a cytospin centrifuge (200 ×g, 5 min). The cells were fixed with 4% paraformaldehyde (PFA) in PBS without Ca2+/Mg2+ for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. For blocking, the cells were incubated in Power Block (Biogenex, Hague, The Netherlands) for 5 min at room temperature. For immunostaining, the cells were incubated for 1 h at room temperature with the following monoclonal antibodies, diluted in blocking solution: MyoD (1:50, Santa Cruz Biotechnology, Heidelberg, Germany), Desmin (1:100, Dako, Hamburg, Germany), and Pax-7 (1:50, R&D Systems). After three washes with wash buffer (1×, Dako), the cells were stained for 30 min at room temperature with Alexa-Fluor-488-labeled goat antimouse antibody (1:100, Invitrogen, Darmstadt, Germany) or Alexa-Fluor 568-conjugated donkey anti-mouse IgG-antibody (1:200, Invitrogen), followed by 15 min of 4′,6-diamidino-2phenylindole dihydrochloride (DAPI) staining (1 μg/ml, Roche, Mannheim, Germany). The cells were mounted with Fluorescence mounting media (Dako), and viewed under a fluorescence microscope (Axiophot epifluorescence microscope, Zeiss, Germany) at a magnification of 200 fold or 400 fold. Pictures were taken with a digital camera (Power Shot G5 digital camera, Canon, Japan). The percentage of cells stained positively for antibody was evaluated by assessing 10 high-power microscopic fields (×400) in each section. Quantitative data were expressed as mean ± SD.
RNA extraction and quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) Total RNA was extracted from MDPCs using the RNeasy mini kit (Qiagen, Hilden Germany) and cDNA was synthesized by the High Capacity RNA-to-cDNA Kit (ABI, Applied Biosystems Inc, Foster City, CA, USA). Synthesized cDNA was analyzed by PCR using the Multiplex PCR Kit (Qiagen). PCR products were separated by electrophoresis on 2% agarose gels. The primers used for PCR were purchased from Eurofins MWG Operon, Ebersberg, Germany (Supplementary Table 2).
Quantitative real-time PCR qRT-PCRs were carried out using Maxima™ SYBR Green qPCR Master Mix (Fermentas, St. Leon-Rot, Germany) and run on a BioRad Chromo 4 (BioRad, München, Germany). Reactions were carried out in triplicates with RT controls, the gene of the ribosomal protein HL32 was used as a reference gene, and data were analyzed using the modified delta Ct method. Primer sequences are listed in Supplementary Table 2.
In vitro muscle cell differentiation The ability of the MDPCs to differentiate into myotubes in vitro was tested by plating them at 1 × 105 cells per well in 6-well plates for 3–5 days in proliferation medium until they reached 70% of confluency. Then, the medium was replaced by muscle fusion medium (MFM: DMEM/F12, 2% FCS, 1% P/S). On days 10–15, acetone fixed cultures were blocked with Power Block then incubated with monoclonal antibodies directed to skeletal myosin heavy chain (MyHC, 1:4000, Abcam, Cambridge, UK) followed by Alexa Flour 568-conjugated donkey anti-mouse IgG (1:100, Invitrogen) for 1 h at room temperature. Negative controls were obtained by omitting the primary antibody. Nuclei were stained with DAPI (1 μg/ml, Roche).
Differentiation assays Differentiation into smooth muscle For smooth muscle differentiation, cells at 70% confluency were switched to DMEM, 15% FCS, and 20 μg/ml endothelial cell growth supplement (Sigma-Aldrich). The control group was grown in a muscle proliferation medium in a humidified atmosphere at 37 °C with 5% CO2. After 10–14 days, cells were fixed in 4% PFA at RT, then incubated with antibodies directed to α-smooth-muscle-actin (1:50, Dako) or Desmin (1:100, Dako) followed by Alexa Flour 488conjugated goat anti-mouse IgG (1:100, Invitrogen), for 30 min at room temperature. Negative controls were obtained by omitting primary antibodies. Nuclei were stained with DAPI (1 μg/ml, Roche).
Differentiation into adipocytes To induce adipogenesis, adipogenic induction medium was prepared with DMEM supplemented with 1 mM dexamethasone, 10 mM isobutylmethylxanthine, 100 mM indomethacin, and 10 μg/ml insulin (all from Sigma-Aldrich), 10% FBS, 1% (P/S). MDPCs were plated at a density of 4 × 104 cells/cm2 of plastic culture flasks and incubated in a humidified atmosphere at 37 °C with 5% CO2. After confluency was reached, the adipocyte induction medium was changed every three days continuously for two to three weeks. The control group was grown in muscle proliferation medium. Oil Red O (Sigma-Aldrich) was used as a histological stain to visualize the presence of lipid droplets as a hallmark of adipocytes.
Differentiation into osteoblasts To induce osteogenesis, osteogenic induction medium was prepared by supplementing DMEM with 10% FBS, 50 mg/ml L-ascorbic acid (Merck KGaA, Munich, Germany), 10 mM-glycerophosphate (Sigma-Aldrich), 100 nM dexamethasone (SigmaAldrich), and 1% P/S. MDPCs were plated at a density of 2.4 × 104 cells/cm2 of plastic culture flasks and incubated in osteogenic induction medium in a humidified atmosphere at 37 °C with 5% CO2. The medium was changed every three days continuously for two to three weeks. The control group was grown in muscle proliferation medium. Alizarin Red S (pH 4.2, Sigma-Aldrich) was used to stain matrix mineralization associated with osteoblasts.
Clonogenic assay By FACS sorting, cells were plated on ultra low-adhesion 96-well plates on the basis of both ALDH1 activity and expression of CD56
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at a single cell per well. Wells containing either none or more than one cell were excluded for further analysis. Cells were cultivated in a 1:1 mixture of fresh and conditioned MPM and MFM at humidified atmosphere of 5% CO2 for 2–3 weeks. The clone formation was observed by inverted phase contrast microscopy (Axiovert 40CFL, Zeiss).
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and isotype-matched control antibodies. Cell sorting was performed on an Aria cell sorter (BD Biosciences).
Results Isolation and in vitro expansion of myosphere-generating cells
Flow cytometry phenotyping and sorting analyses The identification of ALDH1 activity from MDPCs was conducted by using the Aldefluor assay (Stem Cell Technologies, Durham, NC, USA). MDPCs were collected using a 40 μm mesh and disaggregated into single cells by Trypsin/EDTA digestion for 10 min and followed by 20 times up- and down pipetting using a 1000 μl pipette tip. The single cell suspension was washed twice in PBS buffer then it was suspended in Aldefluor assay buffer and incubated for 40 min at 37 °C. As a negative control, for each sample of cells an aliquot was treated with 50 mmol/l diethylaminobenzaldehyde (DEAB, Stem Cell Technologies), a specific ALDH1 inhibitor. Next, for cell surface antigen phenotyping, cells were resuspended in 100 μl Aldefluor incubation buffer and then stained with 20 μl anti-CD34-PE, 20 μl anti-CD56-APC, 20 μl anti-CD45-FITC, and 5 μl 7-AAD (all antibodies from BD bioscience, San Jose, CA, USA) per 106 cells. The cells were incubated at 4 °C for 15 min in the dark. Following incubation, cells were centrifuged, washed twice and resuspended in Aldefluor kit buffer and analyzed by flowcytometry (FACS-Calibur, Becton-Dickinson, Heidelberg, Germany) using the Cell Quest-Pro software. Typically 104 cells were acquired and analyzed. For FACS sorting, cells were resuspended in PBS buffer at 1 × 107 cells/ml and populations were sorted on the basis of both ALDH1 activity and expression of CD56. The sorting gates were established, using as negative controls the cells treated with DEAB
Fresh biopsies of skeletal muscle were immediately processed to obtain single cell suspensions. For the first 48 h, the nutrient medium contained a mixture of cellular debris and a few small rounded cells suspended in the medium. Within 3–4 days, some of the cells aggregated into clusters of 10–20 cells. During the next few days, these myospheres continued to grow in size and number. Cells proliferated to form floating myospheres in cell suspension after 7–10 days (Figs. 1A and B). Emerging myospheres appeared spherical, had a smooth regular surface and appeared shiny in phase contrast microscopy. When culture conditions were suboptimal, or the cells had been mechanically damaged the myospheres became irregular, contained dark regions, vacuoles, and dead cells. At that time point the spheroids had reached a maximum diameter and did not further expand. One could hypothesize that this is probably a result from a cessation of cell division either because of continued differentiation, or degeneration of core stem cells due to limited diffusion of nutrients or oxygen. According to our observation, we were able to generate more progenitor stem cells, when the myospheres were dissociated and passaged before this stage. These myospheres could be maintained, by serial passages, as suspended myospheres, for at least 5 months without losing their proliferative capacity, indicating self-renewal capacity. When myospheres were grown in collagen-coated flasks 80% of them attached rapidly within 24 h. After 4–5 days, more than 80% of myospheres adhered to the bottom and started to grow
Fig. 1 – Phase contrast microscopy of myosphere cultures. Suspension cultures of myospheres at day 4 (A) and day 7 (B) are shown. Radial outgrowth of myogenic cells from adherent myospheres appeared at day 2 (C) and 5 (D).
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out either as rounded or spindle-shaped cells (Figs. 1C and D). Numerous processes radiated from the initial colony, cells migrated outwards in all directions, and individual, phase-bright cells migrated away from the main mass of the myosphere. These “sun-like”-shaped myospheres, over time, gradually disappeared.
Proliferation of MDPCs To examine the proliferative capacity, MDPCs were seeded in MPM and passaged every 5–7 days, when the cells reached 60% of confluence. We observed that the MDPCs could be passaged at least 18 times during 20 weeks of in vitro culture. During this period, they maintained myogenic potential, which was demonstrated by a preserved ability to differentiate and express Desmin (data not shown). MDPCs could undergo more than 40 PDs before the onset of senescence. Growth was slow in the first 6 weeks with a PDT of 171 ± 2.6 h. We observed the fastest growth rate during weeks 5–16 (passages 4–12) with a PDT of 60 ± 3.4 h. After around 17 weeks of culture, cell growth slowed down to a PDT of 112 ± 1.8 h. Supplementary Fig. 1 illustrates the cumulative number of population doublings (PDs) during 20 weeks of continuous MDPCs culture of three human muscle samples. Five of the 20 samples did not expand in vitro due to either bacterial contamination (2 cases) or a critically small starting weight of the muscle biopsy (3 cases). In our hands, none of the successful cultures had a starting weight below 100 mg. In the tested population, sex or donor age seemed not to be critical (Supplementary Table 1).
Phenotypic characterization of myospheres Myospheres contained cells that were strongly immunoreactive for stem cell- and progenitor cell markers but not for markers of differentiated muscle. Examination of freshly isolated myospheres revealed that most of the colonies expressed Pax7,
suggesting their origin from satellite cells (Figs. 2A–C). Cells in myospheres also expressed ALDH1, recently identified as a marker for a population of human skeletal muscle progenitors [14] (Figs. 2D–F).
Clonogenicity of MDPCs To verify that a single myosphere-derived cell has the capacity to generate a myosphere, we initiated a series of single-cell-cloning experiments using single FACS-sorted MDPCs with expression of CD56 and ALDH1 activity. Colonies developed in 4.2% of the wells (6 clones found of 144 wells, Figs. 3A–D) and their developmental potential was tested after 2 to 3 weeks. As detailed in Supplementary Fig. 3, cloned cells cultured in MPM and MFM differentiated into myogenic cells expressing Desmin. These cells also fused to become multinucleated muscle fibers expressing MyHC.
Gene expression analysis of stem cell markers The continuous expression of stem cell-relevant marker genes in cultured MDPCs was investigated by RT-PCR. After the 3rd, 6th, and 9th passage, MDPCs showed sustained expression of Pax7, Nanog, Sox2, and Oct3/4, genes important for the self-renewal capacity and the maintenance of pluripotency (Supplementary Fig. 2).
Flow cytometric analysis of human muscle biopsies and MDPCs A mononucleated cell suspension dissociated from human muscle biopsies (n = 15) and a single cell suspension generated from MDPCs were analyzed by FACS using CD56, CD45 and CD34 (Fig. 4). In primary cell populations derived of human muscle biopsies 4.58% (±1.04) were positive for CD56, 1.02% (±0.27) for CD45 and 5.34 (±0.38) were expressing CD34, suggesting a proendothelial and hematopoietic commitment of
Fig. 2 – Immunophenotyping of myospheres. (A and D) DAPI (blue) nuclear staining. Myospheres were strongly reactive for (B) Pax7 (green), and (E) ALDH1 (green). (C and F) Overlay of (A and B) and (D and E).
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Fig. 3 – Formation of a myosphere starting from a single cell. Documentation of a culture started with a single cell clone that propagates and subsequently forms a multicellular myosphere in suspension. Pictures were taken at days 1, 4, 7, and 14 (A–D).
a part of this population (Figs. 4A and B). To explore the effects of three dimensional myosphere cultures on these subpopulations, we examined the 3rd passage of MDPCs from these 15 specimens. Flow cytometry demonstrated that MDPCs were
significantly enriched for sub-populations that expressed CD56 (70.89 ± 1.23%). Importantly, these sub-populations were negative for markers of hematological (CD45) and endothelial (CD34) origin (Figs. 4C and D).
Fig. 4 – Comparative FACS-analyses of surface antigen expression in dissociated human muscle cells and 3rd passage MDPCs. MDPCs showed an enrichment for sub-populations that expressed CD56 but no expression of markers of hematological (CD45) and endothelial origin (CD34). (A and B) Antigen expression in the freshly isolated cell suspension. (C and D) Antigen expression of 3rd passage MDPCs.
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ALDH1 activity and CD56-expression in MDPCs
Phenotypic characterization of MDPCs
To determine whether ALDH1 activity was detectable in MDPCs and whether this is associated with CD56 expression, MDPCs were labeled for ALDH1 and CD56, and then analyzed and sorted by flowcytometry. An overlap of 79.9 ± 0.3% of the ALDH1-positive and CD56-positive cell populations was detected (data not shown). Subsequently, for further analysis of stemness-related transcription factors by RT-PCR, MDPCs were sorted according to their ALDH1 activity and expression of CD56 (Figs. 5A and B). Real-time RT-PCR analysis of sorted ALDH1+/CD56+ cells was compared to the ALDH1−/CD56+ and the ALDH1−/CD56−cell fractions (Fig. 5C). In the double-positive fraction significantly increased levels of Pax7 (17.98 ± 0.86 fold), Oct3/4 (6.05 ± 0.26 fold), Sox2 (3.12 ± 0.29 fold), and Nanog (1.94 ± 0.47 fold) over the double negative population were found. The ALDH1−/CD56+ fraction showed only significantly increased Pax7 (15.77 ± 1.77 fold) while the other markers were not increased over the double negative fraction. For fraction ALDH1+/CD56− too few cells were isolated and therefore TF marker expression could not be determined. The expression of the stemness-related TF correlated to the ALDH1 activity. In contrast the Pax7 expression correlated to CD56 expression and was independent of the ALDH1 activity. No significantly increased expression of TF was found in the ALDH1 negative fraction.
To further define the expression pattern and lineage progression of the cultured MDPCs immunostaining was performed using Pax7, Desmin, and MyoD antibodies (Figs. 6A–K). While approximately 70% of the cells expressed Pax7 (Fig. 6B), only 50% of cells expressed Desmin and MyoD (Figs. 6D, F, and G) in the early passage (3rd passage) of MDPCs. More than 70% of cells expressed Myod and Desmin in the 5th passage of MDPCs (Figs. 6I, J, and K). This suggests that the cultured MDPCs contain cells at various stages of muscle lineage progression and that this progression may manifest as a complex heterogeneity.
Spontaneous differentiation of MDPCs into skeletal muscle cells MDPCs were cultured for 3–5 days in MPM, then for 7–10 more days in MFM. Typical myotubes with an elongated shape and three to five nuclei appeared after 8–10 days and developed further with time, enclosing up to 15 nuclei 10 days later (Fig. 7A). All myotubes that developed under these conditions expressed MyHC (Fig. 7B). MDPCs were capable of spontaneous myogenesis, supporting the hypothesis that these cells are capable of commitment to the myogenic lineage.
Fig. 5 – Expression of stemness markers in FACS sorted MDPCs populations. (A) Negative control used for the definition of the gates for cell-sorting. (B) CD56 and ALDH1 expression and gates used for cell-sorting. Definition of gates: P4: CD56-ALDH1+; P5: CD56+ALDH1+; P6: CD56-ALDH1−; and P7:CD56-ALDH1+. (C) Expression of stem cell-related transcription factors by cells sorted according to ALDH1 activity and CD56 surface expression.
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Multi-lineage differentiation potential of MDPCs Culture of skeletal muscle by the myosphere method resulted in an expansion of stem cell-like cells that could sustain propagation
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and differentiation into myoblasts/myocytes. Multi-lineage progenitor cells have been previously identified in the skeletal muscle [27,28]. Next, we investigated if the MDPCs had multi-lineage mesodermal potential when cultured under the appropriate
Fig. 6 – Phenotypic characterization of MDPCs. MDPCs are composed of a heterogeneous cell population, that contains cells at various stages of muscle lineage progression, including progenitor stem cells, satellite cells, myoblasts and myogenic cells. (A and B) Marker expression of Pax7 (green), (C and D) MyoD (green), and (E, F, and G) Desmin (red) in the 3rd passage of the MDPCs. (H, I, J, and K) The expression of Myod (green) and Desmin (red) in the 5th passage of MDPCs. (A, C, E, and H) DAPI (blue) was used for counterstaining of the nuclei.
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Fig. 7 – Spontaneous and multi-lineage differentiation in vitro. (A) MDPCs differentiated into myotubes (phase contrast, the white arrow points to multinucleated myotubes). (B) Immunostaining for MyHC (red). (C) Immunostaining for smooth muscle alpha-actin. (D) Control cells differentiated into myogenic cells stained for Desmin (green). Nuclei were stained with DAPI (blue). (E) Crystal-like extracellular matrix deposits were seen on the surface of most monolayer cells at the end of the incubation period in osteogenic induction medium. (F) Alizarin Red S-staining of calcium phosphate formed by the action of alkaline phosphatase on extracellular matrix (orange–red, at pH4 with Alizarin Red S). (G) Lipid droplets in the cytoplasm of the cells detected by bright-field phase-contrast microscopy. (H) Verification of lipid accumulation by staining with Oil Red O (red). The counterstaining was performed with hematoxylin. At the white arrow can be seen some fat globules within the cells. The globules stain reddish-orange.
differentiation conditions. Therefore, the population of MDPCs was analyzed for their multi-lineage differentiation potential into smooth muscle, osteoblastic, and adipocyte cell lineages.
Smooth muscle cell induction After 10–14 days of incubation in smooth muscle induction and differentiation media, visually distinguishable morphologic differ-
ences between the treated cells and the control cells became obvious. In the presence of smooth muscle induction media, the cells acquired typical smooth muscle morphology with a “hill and valley morphology”, which is a hallmark of smooth muscle cells in vitro. In some regions the cells formed multilayered sheets that overlapped (“hill”), while other regions formed monolayers (“valley”) that together appear as a “hill–valley” shape. Immunostaining with antibodies specific to smooth muscle alpha-actin
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(SMA), an established marker of the smooth muscle lineage, showed a characteristic staining as shown in Fig. 7C. More than half of the cells (54.7 ± 4.1%) were SMA positive. In contrast, the cells did not stain for alpha-SMA protein in the absence of induction media and did not change to the smooth muscle morphology (data not shown). Instead, cells grew until confluency and formed myotube-like structures, which were detected by the expression of Desmin (Fig. 7D).
Osteoblast induction After 7–10 days of incubation in the osteogenic induction medium, the cells started to divide rapidly and to grow to confluency. By phase-contrast microscopy, some crystals, sparsely deposited on the cells became visible. The number of crystals increased with incubation time and became numerous towards the end of the incubation period resulting in a difficulty in identifying the morphology of individual cells (Fig. 7E). The whole monolayer cell culture appeared orange–red after Alizarin Red S staining to visualize the calcium deposits (Fig. 7F). Positive Alizarin Red S staining is indicative of calcium deposition by cells of an osteogenic lineage. As such, it is an early stage marker of matrix mineralization, a crucial step towards the formation of calcified extracellular matrix associated with true bone formation. The percentage of cells positive for Alizarin Red S staining was 32.4 ± 4.1%.
Adipocyte induction To analyze the adipogenic differentiation capacity, cells were cultured for 21 days under conditions designed to stimulate adipogenic differentiation. After 10–14 days of incubation in adipogenic medium, the cells were observed to change into large polygonal cells. During incubation, small vesicles became gradually but progressively visible in the cytoplasm of cells. Over time, these vesicles grew larger in size and number and fused to big droplets (Fig. 7G). At the end of the incubation period, almost 50% (43.3 ±2.7%) of the MDPCs had lipid droplets staining positively with Oil Red O (Fig. 7H). Importantly, the findings in the control groups showed that there were no alterations in morphological characteristics. This group is uniformly composed of the spindle-like myogenic cells. Therefore this differentiation is induced and not occurring spontaneously and there was no contamination with adipocytes present. Taken together MDPC had the potential to propagate and differentiate into multiple mesodermal cell types.
Discussion Given the putative therapeutic potential of adult stem cells, there is a need for an effective technique to isolate, purify, and to expand such cells. Much progress in understanding muscle development and stem cell function has been generated from work in mice. However, at present, only limited knowledge of human muscle development and maintenance is available. In the current study, we show that human neck skeletal muscle stem cells can be easily purified by generating “myospheres” containing MDPCs in suspension culture. These cultures after expansion yield large numbers of differentiation-competent myoblasts. In our study, a different procedure than previously described for isolation and expansion of animal skeletal-muscle stem cells [29] was employed
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to isolate and expand human skeletal-muscle stem cells. Myospheres were cultured in serum-free culture medium containing EGF and bFGF. This approach helped to prevent the growth of contaminating differentiated cells, mainly endothelial cells and fibroblasts, that are also growing when cultivated in suspension under conditions that favor selective growth of stem cells [30]. Furthermore, cell–cell contact and membrane-associated factors, known to be important for the stem cell physiology and propagation could be involved in this sphere culture system [31], which is in accordance with the notion that stem cells will only retain their pluripotency within an appropriate environment, as suggested by the “niche” hypothesis [32]. We here confirmed the existence of putative adult skeletal muscle stem cells/progenitor cells that were capable of propagating over extended periods of time as myospheres with a sustained ability to proliferate and differentiate. We succeeded albeit the size of the human muscle biopsies that were used for our study was limited and they were obtained from patients with an average age above 60 years. In our hands, stem cell cultures could be generated from 15 of the 20 donors. Smaller biopsies generally resulted in lower numbers of MDPCs. We found that cultures from samples weighing less than 100 mg were not successful. Thus, it can be assumed that successful application of the described method requires that a critical number of stem cells have to be contained in the sample. More important is, that the muscle stem cells could be cultured and expanded for 20 weeks or 18 passages even when obtained at an average donor age of 63 (62.5 ± 8.3), demonstrating a sustained self-renewal capacity. A number of individual clones derived from verified single cells were successfully expanded, and several of these clones could differentiate into myogenic cells expressing Desmin and MyHC as markers of muscle fiber differentiation [25]. Since the individual colonies were each obtained from a single cell, this is an evidence of the capacity of self-renewal. MDPCs isolated from adult human skeletal muscle share features of muscle stem cells, which express the stem cell marker CD56, ALDH1, Pax7, Myod, and the myogenic marker Desmin. In concordance with the observation that cells contained in the MDPCs expressed the satellite cell markers CD56 and Pax7, the MDPCs were shown to differentiate into skeletal-muscle fibers. Furthermore, they were also able to give rise to different cell types of mesoderm origin namely smooth muscle cells, adipocytes, and osteogenic cells if grown in the appropriate differentiation media. One important finding of the study was that undifferentiated MDPCs not only showed gene expression of Pax7, but also of the TFs Sox2, Nanog, and Oct3/4, which indicates the existence of multipotent stem cells in MDPCs. Most interestingly, Sox2 and Oct3/4 also play critical roles in inducing pluripotency in adult somatic cells. These cells known as induced pluripotent stem cells (iPSCs) may provide a promising source of patient specific cells for cellular replacement therapies [33–35]. Recent scientific findings discovered that only Oct3/4 is sufficient to reprogram human neural stem cells to iPSCs [36]. Due to the intrinsic expression of these TFs, MDPCs would allow for a much easier and more effective induction of pluripotency by TFs. Furthermore, we showed that cells with elevated expression of the confirmed satellite cell markers CD56 and Pax7 can be divided into two groups according to the ALDH1 activity. The subset of satellite cells that is characterized by the co-expression of ALDH1 exhibits an increased expression of stemness-related TFs. This result shows that the satellite cell population in human muscle is heterogeneous in vitro.
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It is possible that satellite cells coexist with a population of multipotent stem cells that could even be the precursors of satellite cells as indicated by the expression of stemness-related TFs. However, we have not yet determined if these cells constitute a distinct cell lineage or if they arise from the main satellite cell population that resides in a specialized niche. The molecular mechanisms and the biological significance of such phenomena remain to be determined. In our experiments we used short term skeletal muscle cultures, that were subjected to spheroid culture methods in order to enrich for putative muscle stem cells. These spheroids were three-dimensional cultures composed of cells with heterogeneous phenotypes. In these initial studies cloning experiments were not performed to study the full differential potential of these cultures. Although we have investigated the proliferative capacity at a clonal level, we have not yet experimentally and systematically proven the differentiation potential of ALDH1+ cell clones. Initial experiments of cloned ALDH1+ cells sorted from spheroids showed their potential to become myogenic and fused muscle fibers (Supplementary Fig. 3). These experiments could be limited, however, by the fact that according to our observation the growth capacity slows down after 16 weeks or later passages. In future experiments ALDH1+ cells should be directly isolated form fresh skeletal muscle tissue, clonally expanded and investigated for their potential to differentiate. This experiment will also help to clarify if ALDH1+ CD56+ cells are located in the hierarchy of pluripotent muscle stem cells upstream or downstream of ALDH1− CD56+ cells. These questions are currently under investigation in our laboratory. In summary, our investigation demonstrates that MDPCs derived from human skeletal muscle harbor a population of stem cell-like cells that can be further sub-divided with appropriate markers. They have characteristic features of multipotent stem cells, as demonstrated by their ability to differentiate into several mesodermal lineage cell types. Considering the straightforward and reproducible isolation and expansion of a stem cell population, the myosphere culture method may represent an attractive source for myogenic stem cells for research and MDPCs may provide great potential for use in cell-based regenerative therapies.
Acknowledgments We would like to thank Mr. Karym El Sayed and PD Dr. Andreas Haisch for their work in the initial phase of the project. This work was financially supported by funding from the Charité “Leistungsorientierte Förderung” to Dr. Andreas E. Albers and Dr. Katharina Stoelzel.
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.yexcr.2011.01.019.
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Research Article
Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture Yan Wei a,b , Yuan Li b , Chao Chen a , Katharina Stoelzel a , Andreas M. Kaufmann c , Andreas E. Albers a,⁎ a
Department of Otolaryngology, Head and Neck Surgery Charité-Universitätsmedizin Berlin, Berlin, Germany Department of Otolaryngology, Head and Neck Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guang Zhou, China c Clinic for Gynecology CCM/CBF, Charité-Universitätsmedizin Berlin, Berlin, Germany b
A R T I C L E I N F O R M A T I O N
A B S T R A C T
Article Chronology:
Human skeletal muscle contains an accessible adult stem-cell compartment in which
Received 17 September 2010
differentiated myofibers are maintained and replaced by a self-renewing stem cell pool.
Revised version received
Previously, studies using mouse models have established a critical role for resident stem cells in
14 January 2011
skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the
Accepted 15 January 2011
reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate
Available online 27 January 2011
for extended periods of time as floating clusters of rounded cells, termed “myospheres” or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional
Keywords:
properties of these cells were determined using reverse transcription-polymerase chain reaction
Muscular dystrophy
(RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are
Satellite cell
clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem
Myosphere
cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be
Induced differentiation
maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times,
Self-renewal
despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were
Adult stem cell
successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types. © 2011 Elsevier Inc. All rights reserved.
⁎ Corresponding author at: Hals-Nasen-Ohrenklinik, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200, Berlin. E-mail address: [email protected] (A.E. Albers). Abbreviations: MDPCs, myosphere-derived progenitor cells; ALDH1, aldehyde dehydrogenase-1; SCs, satellite cell; TFs, transcription factors; FACS, fluorescence-activated cell sorting; SMA, smooth muscle alpha-actin; iPSCs, induced pluripotent stem cells; MGM, myosphere-growing medium; MPM, muscle proliferation medium; MFM, muscle fusion media; RT-PCR, reverse transcription-polymerase chain reaction; CPD, cumulative population doubling; PDT, population doubling time 0014-4827/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2011.01.019
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Introduction Therapeutic approaches for skeletal muscle degeneration by cell transplantation have been impeded so far by low numbers of accessible cells, poor cellular survival rates, and lack of reproducible methods of expansion [1–4]. Ideal graft cells should be nonimmunogenic, easy to expand in vitro, and be able to engraft and differentiate into functional myotubes. Satellite cells (SCs) are quiescent cells that are located beneath the basal lamina of skeletal muscle fibers. They are recruited for muscle growth, maintenance, repair, and regeneration [5]. So far, difficulties in studying human skeletal muscle arose, not only because of practical difficulties in obtaining muscle biopsies to prepare cells in sufficient quantity, but also due to the lack of markers suitable to distinguish satellite cells from myonuclei and other cells present within the skeletal muscle. That is why most research so far relied on animal studies focusing on rodents, particularly mice. Moreover, directly isolated SCs are of low quantity and an expansion step is highly desirable. The spontaneous formation of floating spheres, by three dimensional aggregation of cells cultivated under ultra-low adhesion conditions, is a known prerogative of neural stem cells, endothelial stem cells, and cardiomyocytes [6–8]. These culture methods imitate the three-dimensional structure of tissue and involve as little manipulation as possible to maintain the cellular differentiation potential. In suspension culture, differentiated cells stop dividing and die whereas stem cells continue to proliferate in an apparently asymmetric way by giving rise to many secondary spheres with an exponential growth rate [9]. Recent research demonstrated that muscle stem cells could be isolated and expanded from mice by forming myospheres, which have the potential capability of selfrenewal and multi-lineage differentiation [10,11]. However, to our knowledge, no similar stem cell research involving an isolation and expansion step has been reported concerning human skeletal muscle. So far, only CD56 and Pax7 have been extensively used for identification of SCs on sections of human muscle [12,13]. More recently, Vauchez and colleagues demonstrated that aldehyde dehydrogenase (ALDH) activity was a novel marker for a population of human skeletal muscle progenitors [14]. Investigations confirmed that the SCs are a heterogeneous cell population and only a subset of SCs exhibited asymmetric cell division, longterm Bromodeoxyuridine (BrdU) retention, and appear to be the progenitors of satellite cells [15–19]. It was reported that Oct4, Sox2, and Nanog, which form a self-organized core of transcription factors (TFs), maintain pluripotency and self-renewal capacity of embryonic stem cells [20,21]. More recently, expression of these TFs in adult stem cells suggests, that these stem cells are farther upstream in the line of differentiation [22–24]. Nevertheless, until now, nothing has been reported about the relationship between these TFs and human muscle stem cells. In this study, we prospectively isolated and expanded cells by generating “myospheres” that consist of myosphere-derived progenitor cells (MDPCs) from human skeletal muscle samples. MDPCs maintain the capacity of self-renewal, proliferation and multipotentiality. Our studies also indicate that heterogeneity in the expression of molecular markers in MDPCs may reflect a functional diversity within these cell populations. This observation is further supported by differential expression of TFs. Considering their unproblematic isolation and their proliferative potential, MDPCs could have a significant impact on future clinical strategies to treat patients with muscular disease.
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Materials and methods Human skeletal muscle Skeletal muscle biopsies were obtained from the omohyoid muscle of 20 patients (Supplementary Table 1), who underwent surgery at the Department for Otolaryngology, Head and Neck surgery, Campus Benjamin Franklin, Charité, Universitätsmedizin, Berlin, Germany. Most patients underwent surgery for the resection of cervical lymphnodes as indicated by the presence of a head and neck carcinoma. During this surgery the omohyoid muscle is often cut for optimal surgical exposure. Biopsies were typically taken from one of the free ends. To exclude any infiltration of the muscle tissues, only those cases were selected, where the resection margins were free of cancer as stated by the pathologist. The patients were free of known muscular pathologies. The study was carried out with the approval of the institutional ethics committee (IRB #EA4/033/09). Informed consent was obtained from each patient whose tissues were collected for the study.
Isolation of adult human muscle stem cells Skeletal-muscle stem cells were isolated from the muscle biopsies (0.1–1 g) by a procedure similar to that previously described for human muscle [25]. Briefly, the skeletal muscle tissue was minced into 1–3 mm3 pieces and washed with phosphate buffered saline without Ca2+/Mg2+ (PBS) several times to remove fat and contaminating red blood cells. Muscle biopsies were then finely minced and digested for 60 to 120 min at 37 °C with a mixture of type-I and type-IV collagenase (100 μg/ml) and dispase (1.2 μg/ml; all from GIBCO, Invitrogen, Darmstadt, Germany). The digested tissue was mechanically dissociated and then passed through a 40 μm mesh to obtain a single cell suspension. The single cell suspensions were plated in Corning* Ultra-Low Attachment 6 well plates (Fisher Scientific, Loughborough, UK) containing a myosphere-growing medium (MGM: DMEM/F12, EGF 20 ng/ml, bFGF 20 ng/ml) and kept at humidified atmosphere of 5% CO2 in air.
Myosphere formation and expansion Fresh MGM was added to the cultures every second day (500 μl per well). After 5 days, the suspension was transferred into 50 ml conical tubes and the cell clusters were allowed to sediment for 30 min. Then half of the medium was replaced with new MGM and the cells were replated. After 7–10 days cells proliferated to form floating myospheres. Myospheres were picked with a 40 μm mesh from the media and transferred into a collagen type I (SigmaAldrich, Munich, Germany) coated culture flask in muscle proliferation media (MPM: DMEM/F12, 20% FCS, bFGF 5 ng/ml, 1% P/S). When the cultures approached 60% confluency the MDPCs were digested and passaged at a ratio of 1:2.
Growth rate To evaluate the growth rate of MDPCs, cells were seeded at 3000 cells/cm2 in 25 cm2 tissue culture flasks (Sarsted, Numbrecht, Germany) in MPM. Cells were detached using 0.25% trypsin/EDTA (Biochrom AG, Berlin, Germany) every 5–10 days and the cell
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number was determined. This procedure was repeated over a period of 20 weeks. The calculated population doublings were summarized to compute the cumulative population doubling (CPD) for each of the three cell samples. We calculated the population doubling time (PDT) as previously described [26] by calculating PDT = T/(log2(NF/N0)), where T = time in culture, N0 = initial number of cells, and NF = final number of cells.
Immunocytochemistry Adherent cells from passage 3, 5, 6, 8 and 10 were grown on lysine (0.1 mg/ml, Biochrom AG) coated glass coverslips respectively in MPM. Intact myospheres were put on a glass slide using a cytospin centrifuge (200 ×g, 5 min). The cells were fixed with 4% paraformaldehyde (PFA) in PBS without Ca2+/Mg2+ for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. For blocking, the cells were incubated in Power Block (Biogenex, Hague, The Netherlands) for 5 min at room temperature. For immunostaining, the cells were incubated for 1 h at room temperature with the following monoclonal antibodies, diluted in blocking solution: MyoD (1:50, Santa Cruz Biotechnology, Heidelberg, Germany), Desmin (1:100, Dako, Hamburg, Germany), and Pax-7 (1:50, R&D Systems). After three washes with wash buffer (1×, Dako), the cells were stained for 30 min at room temperature with Alexa-Fluor-488-labeled goat antimouse antibody (1:100, Invitrogen, Darmstadt, Germany) or Alexa-Fluor 568-conjugated donkey anti-mouse IgG-antibody (1:200, Invitrogen), followed by 15 min of 4′,6-diamidino-2phenylindole dihydrochloride (DAPI) staining (1 μg/ml, Roche, Mannheim, Germany). The cells were mounted with Fluorescence mounting media (Dako), and viewed under a fluorescence microscope (Axiophot epifluorescence microscope, Zeiss, Germany) at a magnification of 200 fold or 400 fold. Pictures were taken with a digital camera (Power Shot G5 digital camera, Canon, Japan). The percentage of cells stained positively for antibody was evaluated by assessing 10 high-power microscopic fields (×400) in each section. Quantitative data were expressed as mean ± SD.
RNA extraction and quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) Total RNA was extracted from MDPCs using the RNeasy mini kit (Qiagen, Hilden Germany) and cDNA was synthesized by the High Capacity RNA-to-cDNA Kit (ABI, Applied Biosystems Inc, Foster City, CA, USA). Synthesized cDNA was analyzed by PCR using the Multiplex PCR Kit (Qiagen). PCR products were separated by electrophoresis on 2% agarose gels. The primers used for PCR were purchased from Eurofins MWG Operon, Ebersberg, Germany (Supplementary Table 2).
Quantitative real-time PCR qRT-PCRs were carried out using Maxima™ SYBR Green qPCR Master Mix (Fermentas, St. Leon-Rot, Germany) and run on a BioRad Chromo 4 (BioRad, München, Germany). Reactions were carried out in triplicates with RT controls, the gene of the ribosomal protein HL32 was used as a reference gene, and data were analyzed using the modified delta Ct method. Primer sequences are listed in Supplementary Table 2.
In vitro muscle cell differentiation The ability of the MDPCs to differentiate into myotubes in vitro was tested by plating them at 1 × 105 cells per well in 6-well plates for 3–5 days in proliferation medium until they reached 70% of confluency. Then, the medium was replaced by muscle fusion medium (MFM: DMEM/F12, 2% FCS, 1% P/S). On days 10–15, acetone fixed cultures were blocked with Power Block then incubated with monoclonal antibodies directed to skeletal myosin heavy chain (MyHC, 1:4000, Abcam, Cambridge, UK) followed by Alexa Flour 568-conjugated donkey anti-mouse IgG (1:100, Invitrogen) for 1 h at room temperature. Negative controls were obtained by omitting the primary antibody. Nuclei were stained with DAPI (1 μg/ml, Roche).
Differentiation assays Differentiation into smooth muscle For smooth muscle differentiation, cells at 70% confluency were switched to DMEM, 15% FCS, and 20 μg/ml endothelial cell growth supplement (Sigma-Aldrich). The control group was grown in a muscle proliferation medium in a humidified atmosphere at 37 °C with 5% CO2. After 10–14 days, cells were fixed in 4% PFA at RT, then incubated with antibodies directed to α-smooth-muscle-actin (1:50, Dako) or Desmin (1:100, Dako) followed by Alexa Flour 488conjugated goat anti-mouse IgG (1:100, Invitrogen), for 30 min at room temperature. Negative controls were obtained by omitting primary antibodies. Nuclei were stained with DAPI (1 μg/ml, Roche).
Differentiation into adipocytes To induce adipogenesis, adipogenic induction medium was prepared with DMEM supplemented with 1 mM dexamethasone, 10 mM isobutylmethylxanthine, 100 mM indomethacin, and 10 μg/ml insulin (all from Sigma-Aldrich), 10% FBS, 1% (P/S). MDPCs were plated at a density of 4 × 104 cells/cm2 of plastic culture flasks and incubated in a humidified atmosphere at 37 °C with 5% CO2. After confluency was reached, the adipocyte induction medium was changed every three days continuously for two to three weeks. The control group was grown in muscle proliferation medium. Oil Red O (Sigma-Aldrich) was used as a histological stain to visualize the presence of lipid droplets as a hallmark of adipocytes.
Differentiation into osteoblasts To induce osteogenesis, osteogenic induction medium was prepared by supplementing DMEM with 10% FBS, 50 mg/ml L-ascorbic acid (Merck KGaA, Munich, Germany), 10 mM-glycerophosphate (Sigma-Aldrich), 100 nM dexamethasone (SigmaAldrich), and 1% P/S. MDPCs were plated at a density of 2.4 × 104 cells/cm2 of plastic culture flasks and incubated in osteogenic induction medium in a humidified atmosphere at 37 °C with 5% CO2. The medium was changed every three days continuously for two to three weeks. The control group was grown in muscle proliferation medium. Alizarin Red S (pH 4.2, Sigma-Aldrich) was used to stain matrix mineralization associated with osteoblasts.
Clonogenic assay By FACS sorting, cells were plated on ultra low-adhesion 96-well plates on the basis of both ALDH1 activity and expression of CD56
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at a single cell per well. Wells containing either none or more than one cell were excluded for further analysis. Cells were cultivated in a 1:1 mixture of fresh and conditioned MPM and MFM at humidified atmosphere of 5% CO2 for 2–3 weeks. The clone formation was observed by inverted phase contrast microscopy (Axiovert 40CFL, Zeiss).
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and isotype-matched control antibodies. Cell sorting was performed on an Aria cell sorter (BD Biosciences).
Results Isolation and in vitro expansion of myosphere-generating cells
Flow cytometry phenotyping and sorting analyses The identification of ALDH1 activity from MDPCs was conducted by using the Aldefluor assay (Stem Cell Technologies, Durham, NC, USA). MDPCs were collected using a 40 μm mesh and disaggregated into single cells by Trypsin/EDTA digestion for 10 min and followed by 20 times up- and down pipetting using a 1000 μl pipette tip. The single cell suspension was washed twice in PBS buffer then it was suspended in Aldefluor assay buffer and incubated for 40 min at 37 °C. As a negative control, for each sample of cells an aliquot was treated with 50 mmol/l diethylaminobenzaldehyde (DEAB, Stem Cell Technologies), a specific ALDH1 inhibitor. Next, for cell surface antigen phenotyping, cells were resuspended in 100 μl Aldefluor incubation buffer and then stained with 20 μl anti-CD34-PE, 20 μl anti-CD56-APC, 20 μl anti-CD45-FITC, and 5 μl 7-AAD (all antibodies from BD bioscience, San Jose, CA, USA) per 106 cells. The cells were incubated at 4 °C for 15 min in the dark. Following incubation, cells were centrifuged, washed twice and resuspended in Aldefluor kit buffer and analyzed by flowcytometry (FACS-Calibur, Becton-Dickinson, Heidelberg, Germany) using the Cell Quest-Pro software. Typically 104 cells were acquired and analyzed. For FACS sorting, cells were resuspended in PBS buffer at 1 × 107 cells/ml and populations were sorted on the basis of both ALDH1 activity and expression of CD56. The sorting gates were established, using as negative controls the cells treated with DEAB
Fresh biopsies of skeletal muscle were immediately processed to obtain single cell suspensions. For the first 48 h, the nutrient medium contained a mixture of cellular debris and a few small rounded cells suspended in the medium. Within 3–4 days, some of the cells aggregated into clusters of 10–20 cells. During the next few days, these myospheres continued to grow in size and number. Cells proliferated to form floating myospheres in cell suspension after 7–10 days (Figs. 1A and B). Emerging myospheres appeared spherical, had a smooth regular surface and appeared shiny in phase contrast microscopy. When culture conditions were suboptimal, or the cells had been mechanically damaged the myospheres became irregular, contained dark regions, vacuoles, and dead cells. At that time point the spheroids had reached a maximum diameter and did not further expand. One could hypothesize that this is probably a result from a cessation of cell division either because of continued differentiation, or degeneration of core stem cells due to limited diffusion of nutrients or oxygen. According to our observation, we were able to generate more progenitor stem cells, when the myospheres were dissociated and passaged before this stage. These myospheres could be maintained, by serial passages, as suspended myospheres, for at least 5 months without losing their proliferative capacity, indicating self-renewal capacity. When myospheres were grown in collagen-coated flasks 80% of them attached rapidly within 24 h. After 4–5 days, more than 80% of myospheres adhered to the bottom and started to grow
Fig. 1 – Phase contrast microscopy of myosphere cultures. Suspension cultures of myospheres at day 4 (A) and day 7 (B) are shown. Radial outgrowth of myogenic cells from adherent myospheres appeared at day 2 (C) and 5 (D).
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out either as rounded or spindle-shaped cells (Figs. 1C and D). Numerous processes radiated from the initial colony, cells migrated outwards in all directions, and individual, phase-bright cells migrated away from the main mass of the myosphere. These “sun-like”-shaped myospheres, over time, gradually disappeared.
Proliferation of MDPCs To examine the proliferative capacity, MDPCs were seeded in MPM and passaged every 5–7 days, when the cells reached 60% of confluence. We observed that the MDPCs could be passaged at least 18 times during 20 weeks of in vitro culture. During this period, they maintained myogenic potential, which was demonstrated by a preserved ability to differentiate and express Desmin (data not shown). MDPCs could undergo more than 40 PDs before the onset of senescence. Growth was slow in the first 6 weeks with a PDT of 171 ± 2.6 h. We observed the fastest growth rate during weeks 5–16 (passages 4–12) with a PDT of 60 ± 3.4 h. After around 17 weeks of culture, cell growth slowed down to a PDT of 112 ± 1.8 h. Supplementary Fig. 1 illustrates the cumulative number of population doublings (PDs) during 20 weeks of continuous MDPCs culture of three human muscle samples. Five of the 20 samples did not expand in vitro due to either bacterial contamination (2 cases) or a critically small starting weight of the muscle biopsy (3 cases). In our hands, none of the successful cultures had a starting weight below 100 mg. In the tested population, sex or donor age seemed not to be critical (Supplementary Table 1).
Phenotypic characterization of myospheres Myospheres contained cells that were strongly immunoreactive for stem cell- and progenitor cell markers but not for markers of differentiated muscle. Examination of freshly isolated myospheres revealed that most of the colonies expressed Pax7,
suggesting their origin from satellite cells (Figs. 2A–C). Cells in myospheres also expressed ALDH1, recently identified as a marker for a population of human skeletal muscle progenitors [14] (Figs. 2D–F).
Clonogenicity of MDPCs To verify that a single myosphere-derived cell has the capacity to generate a myosphere, we initiated a series of single-cell-cloning experiments using single FACS-sorted MDPCs with expression of CD56 and ALDH1 activity. Colonies developed in 4.2% of the wells (6 clones found of 144 wells, Figs. 3A–D) and their developmental potential was tested after 2 to 3 weeks. As detailed in Supplementary Fig. 3, cloned cells cultured in MPM and MFM differentiated into myogenic cells expressing Desmin. These cells also fused to become multinucleated muscle fibers expressing MyHC.
Gene expression analysis of stem cell markers The continuous expression of stem cell-relevant marker genes in cultured MDPCs was investigated by RT-PCR. After the 3rd, 6th, and 9th passage, MDPCs showed sustained expression of Pax7, Nanog, Sox2, and Oct3/4, genes important for the self-renewal capacity and the maintenance of pluripotency (Supplementary Fig. 2).
Flow cytometric analysis of human muscle biopsies and MDPCs A mononucleated cell suspension dissociated from human muscle biopsies (n = 15) and a single cell suspension generated from MDPCs were analyzed by FACS using CD56, CD45 and CD34 (Fig. 4). In primary cell populations derived of human muscle biopsies 4.58% (±1.04) were positive for CD56, 1.02% (±0.27) for CD45 and 5.34 (±0.38) were expressing CD34, suggesting a proendothelial and hematopoietic commitment of
Fig. 2 – Immunophenotyping of myospheres. (A and D) DAPI (blue) nuclear staining. Myospheres were strongly reactive for (B) Pax7 (green), and (E) ALDH1 (green). (C and F) Overlay of (A and B) and (D and E).
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Fig. 3 – Formation of a myosphere starting from a single cell. Documentation of a culture started with a single cell clone that propagates and subsequently forms a multicellular myosphere in suspension. Pictures were taken at days 1, 4, 7, and 14 (A–D).
a part of this population (Figs. 4A and B). To explore the effects of three dimensional myosphere cultures on these subpopulations, we examined the 3rd passage of MDPCs from these 15 specimens. Flow cytometry demonstrated that MDPCs were
significantly enriched for sub-populations that expressed CD56 (70.89 ± 1.23%). Importantly, these sub-populations were negative for markers of hematological (CD45) and endothelial (CD34) origin (Figs. 4C and D).
Fig. 4 – Comparative FACS-analyses of surface antigen expression in dissociated human muscle cells and 3rd passage MDPCs. MDPCs showed an enrichment for sub-populations that expressed CD56 but no expression of markers of hematological (CD45) and endothelial origin (CD34). (A and B) Antigen expression in the freshly isolated cell suspension. (C and D) Antigen expression of 3rd passage MDPCs.
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ALDH1 activity and CD56-expression in MDPCs
Phenotypic characterization of MDPCs
To determine whether ALDH1 activity was detectable in MDPCs and whether this is associated with CD56 expression, MDPCs were labeled for ALDH1 and CD56, and then analyzed and sorted by flowcytometry. An overlap of 79.9 ± 0.3% of the ALDH1-positive and CD56-positive cell populations was detected (data not shown). Subsequently, for further analysis of stemness-related transcription factors by RT-PCR, MDPCs were sorted according to their ALDH1 activity and expression of CD56 (Figs. 5A and B). Real-time RT-PCR analysis of sorted ALDH1+/CD56+ cells was compared to the ALDH1−/CD56+ and the ALDH1−/CD56−cell fractions (Fig. 5C). In the double-positive fraction significantly increased levels of Pax7 (17.98 ± 0.86 fold), Oct3/4 (6.05 ± 0.26 fold), Sox2 (3.12 ± 0.29 fold), and Nanog (1.94 ± 0.47 fold) over the double negative population were found. The ALDH1−/CD56+ fraction showed only significantly increased Pax7 (15.77 ± 1.77 fold) while the other markers were not increased over the double negative fraction. For fraction ALDH1+/CD56− too few cells were isolated and therefore TF marker expression could not be determined. The expression of the stemness-related TF correlated to the ALDH1 activity. In contrast the Pax7 expression correlated to CD56 expression and was independent of the ALDH1 activity. No significantly increased expression of TF was found in the ALDH1 negative fraction.
To further define the expression pattern and lineage progression of the cultured MDPCs immunostaining was performed using Pax7, Desmin, and MyoD antibodies (Figs. 6A–K). While approximately 70% of the cells expressed Pax7 (Fig. 6B), only 50% of cells expressed Desmin and MyoD (Figs. 6D, F, and G) in the early passage (3rd passage) of MDPCs. More than 70% of cells expressed Myod and Desmin in the 5th passage of MDPCs (Figs. 6I, J, and K). This suggests that the cultured MDPCs contain cells at various stages of muscle lineage progression and that this progression may manifest as a complex heterogeneity.
Spontaneous differentiation of MDPCs into skeletal muscle cells MDPCs were cultured for 3–5 days in MPM, then for 7–10 more days in MFM. Typical myotubes with an elongated shape and three to five nuclei appeared after 8–10 days and developed further with time, enclosing up to 15 nuclei 10 days later (Fig. 7A). All myotubes that developed under these conditions expressed MyHC (Fig. 7B). MDPCs were capable of spontaneous myogenesis, supporting the hypothesis that these cells are capable of commitment to the myogenic lineage.
Fig. 5 – Expression of stemness markers in FACS sorted MDPCs populations. (A) Negative control used for the definition of the gates for cell-sorting. (B) CD56 and ALDH1 expression and gates used for cell-sorting. Definition of gates: P4: CD56-ALDH1+; P5: CD56+ALDH1+; P6: CD56-ALDH1−; and P7:CD56-ALDH1+. (C) Expression of stem cell-related transcription factors by cells sorted according to ALDH1 activity and CD56 surface expression.
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Multi-lineage differentiation potential of MDPCs Culture of skeletal muscle by the myosphere method resulted in an expansion of stem cell-like cells that could sustain propagation
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and differentiation into myoblasts/myocytes. Multi-lineage progenitor cells have been previously identified in the skeletal muscle [27,28]. Next, we investigated if the MDPCs had multi-lineage mesodermal potential when cultured under the appropriate
Fig. 6 – Phenotypic characterization of MDPCs. MDPCs are composed of a heterogeneous cell population, that contains cells at various stages of muscle lineage progression, including progenitor stem cells, satellite cells, myoblasts and myogenic cells. (A and B) Marker expression of Pax7 (green), (C and D) MyoD (green), and (E, F, and G) Desmin (red) in the 3rd passage of the MDPCs. (H, I, J, and K) The expression of Myod (green) and Desmin (red) in the 5th passage of MDPCs. (A, C, E, and H) DAPI (blue) was used for counterstaining of the nuclei.
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Fig. 7 – Spontaneous and multi-lineage differentiation in vitro. (A) MDPCs differentiated into myotubes (phase contrast, the white arrow points to multinucleated myotubes). (B) Immunostaining for MyHC (red). (C) Immunostaining for smooth muscle alpha-actin. (D) Control cells differentiated into myogenic cells stained for Desmin (green). Nuclei were stained with DAPI (blue). (E) Crystal-like extracellular matrix deposits were seen on the surface of most monolayer cells at the end of the incubation period in osteogenic induction medium. (F) Alizarin Red S-staining of calcium phosphate formed by the action of alkaline phosphatase on extracellular matrix (orange–red, at pH4 with Alizarin Red S). (G) Lipid droplets in the cytoplasm of the cells detected by bright-field phase-contrast microscopy. (H) Verification of lipid accumulation by staining with Oil Red O (red). The counterstaining was performed with hematoxylin. At the white arrow can be seen some fat globules within the cells. The globules stain reddish-orange.
differentiation conditions. Therefore, the population of MDPCs was analyzed for their multi-lineage differentiation potential into smooth muscle, osteoblastic, and adipocyte cell lineages.
Smooth muscle cell induction After 10–14 days of incubation in smooth muscle induction and differentiation media, visually distinguishable morphologic differ-
ences between the treated cells and the control cells became obvious. In the presence of smooth muscle induction media, the cells acquired typical smooth muscle morphology with a “hill and valley morphology”, which is a hallmark of smooth muscle cells in vitro. In some regions the cells formed multilayered sheets that overlapped (“hill”), while other regions formed monolayers (“valley”) that together appear as a “hill–valley” shape. Immunostaining with antibodies specific to smooth muscle alpha-actin
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(SMA), an established marker of the smooth muscle lineage, showed a characteristic staining as shown in Fig. 7C. More than half of the cells (54.7 ± 4.1%) were SMA positive. In contrast, the cells did not stain for alpha-SMA protein in the absence of induction media and did not change to the smooth muscle morphology (data not shown). Instead, cells grew until confluency and formed myotube-like structures, which were detected by the expression of Desmin (Fig. 7D).
Osteoblast induction After 7–10 days of incubation in the osteogenic induction medium, the cells started to divide rapidly and to grow to confluency. By phase-contrast microscopy, some crystals, sparsely deposited on the cells became visible. The number of crystals increased with incubation time and became numerous towards the end of the incubation period resulting in a difficulty in identifying the morphology of individual cells (Fig. 7E). The whole monolayer cell culture appeared orange–red after Alizarin Red S staining to visualize the calcium deposits (Fig. 7F). Positive Alizarin Red S staining is indicative of calcium deposition by cells of an osteogenic lineage. As such, it is an early stage marker of matrix mineralization, a crucial step towards the formation of calcified extracellular matrix associated with true bone formation. The percentage of cells positive for Alizarin Red S staining was 32.4 ± 4.1%.
Adipocyte induction To analyze the adipogenic differentiation capacity, cells were cultured for 21 days under conditions designed to stimulate adipogenic differentiation. After 10–14 days of incubation in adipogenic medium, the cells were observed to change into large polygonal cells. During incubation, small vesicles became gradually but progressively visible in the cytoplasm of cells. Over time, these vesicles grew larger in size and number and fused to big droplets (Fig. 7G). At the end of the incubation period, almost 50% (43.3 ±2.7%) of the MDPCs had lipid droplets staining positively with Oil Red O (Fig. 7H). Importantly, the findings in the control groups showed that there were no alterations in morphological characteristics. This group is uniformly composed of the spindle-like myogenic cells. Therefore this differentiation is induced and not occurring spontaneously and there was no contamination with adipocytes present. Taken together MDPC had the potential to propagate and differentiate into multiple mesodermal cell types.
Discussion Given the putative therapeutic potential of adult stem cells, there is a need for an effective technique to isolate, purify, and to expand such cells. Much progress in understanding muscle development and stem cell function has been generated from work in mice. However, at present, only limited knowledge of human muscle development and maintenance is available. In the current study, we show that human neck skeletal muscle stem cells can be easily purified by generating “myospheres” containing MDPCs in suspension culture. These cultures after expansion yield large numbers of differentiation-competent myoblasts. In our study, a different procedure than previously described for isolation and expansion of animal skeletal-muscle stem cells [29] was employed
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to isolate and expand human skeletal-muscle stem cells. Myospheres were cultured in serum-free culture medium containing EGF and bFGF. This approach helped to prevent the growth of contaminating differentiated cells, mainly endothelial cells and fibroblasts, that are also growing when cultivated in suspension under conditions that favor selective growth of stem cells [30]. Furthermore, cell–cell contact and membrane-associated factors, known to be important for the stem cell physiology and propagation could be involved in this sphere culture system [31], which is in accordance with the notion that stem cells will only retain their pluripotency within an appropriate environment, as suggested by the “niche” hypothesis [32]. We here confirmed the existence of putative adult skeletal muscle stem cells/progenitor cells that were capable of propagating over extended periods of time as myospheres with a sustained ability to proliferate and differentiate. We succeeded albeit the size of the human muscle biopsies that were used for our study was limited and they were obtained from patients with an average age above 60 years. In our hands, stem cell cultures could be generated from 15 of the 20 donors. Smaller biopsies generally resulted in lower numbers of MDPCs. We found that cultures from samples weighing less than 100 mg were not successful. Thus, it can be assumed that successful application of the described method requires that a critical number of stem cells have to be contained in the sample. More important is, that the muscle stem cells could be cultured and expanded for 20 weeks or 18 passages even when obtained at an average donor age of 63 (62.5 ± 8.3), demonstrating a sustained self-renewal capacity. A number of individual clones derived from verified single cells were successfully expanded, and several of these clones could differentiate into myogenic cells expressing Desmin and MyHC as markers of muscle fiber differentiation [25]. Since the individual colonies were each obtained from a single cell, this is an evidence of the capacity of self-renewal. MDPCs isolated from adult human skeletal muscle share features of muscle stem cells, which express the stem cell marker CD56, ALDH1, Pax7, Myod, and the myogenic marker Desmin. In concordance with the observation that cells contained in the MDPCs expressed the satellite cell markers CD56 and Pax7, the MDPCs were shown to differentiate into skeletal-muscle fibers. Furthermore, they were also able to give rise to different cell types of mesoderm origin namely smooth muscle cells, adipocytes, and osteogenic cells if grown in the appropriate differentiation media. One important finding of the study was that undifferentiated MDPCs not only showed gene expression of Pax7, but also of the TFs Sox2, Nanog, and Oct3/4, which indicates the existence of multipotent stem cells in MDPCs. Most interestingly, Sox2 and Oct3/4 also play critical roles in inducing pluripotency in adult somatic cells. These cells known as induced pluripotent stem cells (iPSCs) may provide a promising source of patient specific cells for cellular replacement therapies [33–35]. Recent scientific findings discovered that only Oct3/4 is sufficient to reprogram human neural stem cells to iPSCs [36]. Due to the intrinsic expression of these TFs, MDPCs would allow for a much easier and more effective induction of pluripotency by TFs. Furthermore, we showed that cells with elevated expression of the confirmed satellite cell markers CD56 and Pax7 can be divided into two groups according to the ALDH1 activity. The subset of satellite cells that is characterized by the co-expression of ALDH1 exhibits an increased expression of stemness-related TFs. This result shows that the satellite cell population in human muscle is heterogeneous in vitro.
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It is possible that satellite cells coexist with a population of multipotent stem cells that could even be the precursors of satellite cells as indicated by the expression of stemness-related TFs. However, we have not yet determined if these cells constitute a distinct cell lineage or if they arise from the main satellite cell population that resides in a specialized niche. The molecular mechanisms and the biological significance of such phenomena remain to be determined. In our experiments we used short term skeletal muscle cultures, that were subjected to spheroid culture methods in order to enrich for putative muscle stem cells. These spheroids were three-dimensional cultures composed of cells with heterogeneous phenotypes. In these initial studies cloning experiments were not performed to study the full differential potential of these cultures. Although we have investigated the proliferative capacity at a clonal level, we have not yet experimentally and systematically proven the differentiation potential of ALDH1+ cell clones. Initial experiments of cloned ALDH1+ cells sorted from spheroids showed their potential to become myogenic and fused muscle fibers (Supplementary Fig. 3). These experiments could be limited, however, by the fact that according to our observation the growth capacity slows down after 16 weeks or later passages. In future experiments ALDH1+ cells should be directly isolated form fresh skeletal muscle tissue, clonally expanded and investigated for their potential to differentiate. This experiment will also help to clarify if ALDH1+ CD56+ cells are located in the hierarchy of pluripotent muscle stem cells upstream or downstream of ALDH1− CD56+ cells. These questions are currently under investigation in our laboratory. In summary, our investigation demonstrates that MDPCs derived from human skeletal muscle harbor a population of stem cell-like cells that can be further sub-divided with appropriate markers. They have characteristic features of multipotent stem cells, as demonstrated by their ability to differentiate into several mesodermal lineage cell types. Considering the straightforward and reproducible isolation and expansion of a stem cell population, the myosphere culture method may represent an attractive source for myogenic stem cells for research and MDPCs may provide great potential for use in cell-based regenerative therapies.
Acknowledgments We would like to thank Mr. Karym El Sayed and PD Dr. Andreas Haisch for their work in the initial phase of the project. This work was financially supported by funding from the Charité “Leistungsorientierte Förderung” to Dr. Andreas E. Albers and Dr. Katharina Stoelzel.
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.yexcr.2011.01.019.
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