Adult muscle-derived stem cells engraft and differentiate into insulin-expressing cells in pancreatic islets of diabetic mice
- Violeta Mitutsova†1,
- Wendy Wai Yeng Yeo†1, 2,
- Romain Davaze1,
- Celine Franckhauser1,
- El-Habib Hani1,
- Syahril Abdullah2,
- Patrice Mollard3,
- Marie Schaeffer3,
- Anne Fernandez1Email author and
- Ned J. C. Lamb1Email author
© The Author(s). 2017
Received: 13 February 2017
Accepted: 16 March 2017
Published: 18 April 2017
Pancreatic beta cells are unique effectors in the control of glucose homeostasis and their deficiency results in impaired insulin production leading to severe diabetic diseases. Here, we investigated the potential of a population of nonadherent muscle-derived stem cells (MDSC) from adult mouse muscle to differentiate in vitro into beta cells when transplanted as undifferentiated stem cells in vivo to compensate for beta-cell deficiency.
In vitro, cultured MDSC spontaneously differentiated into insulin-expressing islet-like cell clusters as revealed using MDSC from transgenic mice expressing GFP or mCherry under the control of an insulin promoter. Differentiated clusters of beta-like cells co-expressed insulin with the transcription factors Pdx1, Nkx2.2, Nkx6.1, and MafA, and secreted significant levels of insulin in response to glucose challenges. In vivo, undifferentiated MDSC injected into streptozotocin (STZ)-treated mice engrafted within 48 h specifically to damaged pancreatic islets and were shown to differentiate and express insulin 10–12 days after injection. In addition, injection of MDSC into hyperglycemic diabetic mice reduced their blood glucose levels for 2–4 weeks.
These data show that MDSC are capable of differentiating into mature pancreatic beta islet-like cells, not only upon culture in vitro, but also in vivo after systemic injection in STZ-induced diabetic mouse models. Being nonteratogenic, MDSC can be used directly by systemic injection, and this potential reveals a promising alternative avenue in stem cell-based treatment of beta-cell deficiencies.
KeywordsMuscle stem cells Beta-cell differentiation Insulin secretion Pancreatic islets
Diabetes mellitus (DM) is identified by excessive blood sugar levels (hyperglycemia) that arises from the incapacity of pancreatic beta cells to supply the hormone insulin (reviewed in  and references therein). Type 1 diabetes mellitus (T1DM) results from autoimmune destruction of pancreatic endocrine beta cells (reviewed in [2–4]), whereas type 2 diabetes mellitus (T2DM) results from progressive exhaustion of beta cells due to acquired insulin resistance . Insulin is secreted by beta cells present in histologically distinct and highly specialized structures in the pancreas—the islets of Langerhans. Pancreatic islets comprise four other cells types: glucagon-producing alpha cells, somatostatin-producing delta cells, pancreatic polypeptide-producing PP cells, and epsilon cells producing ghrelin, with alpha and beta cells representing the majority of islet cells.
The recent identification that stem cells can be derived to undergo lineage-specific differentiation has opened the possibility of stem cell-derived beta-cell replacement therapy in the treatment of T1DM (reviewed in ). Stem cells have been derived in vitro into functional beta cells for transplantation as nanodevices to restore insulin production after implantation either subcutaneously or behind the renal capsule (reviewed in [1, 7]). Beta cells have been obtained from embryonic stems cells (ESC) of both mouse  and human origin  (reviewed in ), or from induced pluripotent stem cells (iPSC) produced through direct transcriptional reprogramming using stemness transcription factor cocktails (reviewed in [10–13]), or through reprogramming via a lineage conversion strategy from fibroblasts . Although still controversial , a number of studies have described techniques for inducing the beta cell-specific reprogramming of fibroblasts of multiple origins into glucose-responsive beta-like cells [16–18]. However, long-term approaches involving differentiation of iPSC and ESC are compromised by the latent potential of residual tumor-promoting stem cells to develop into teratomas or cause tumorigenesis . While these techniques are tedious and time consuming [9, 13], they open the possibility that patient-derived iPSC may ultimately offer a viable source of functional beta cells to overcome the present difficulties in obtaining appropriate pancreas or islet beta-cell donors for transplantation . Indeed in a recent study, differentiated beta cells derived in vitro from human iPSC were shown to secrete insulin in a glucose-responsive manner in vivo when transplanted and encapsulated in immunodeficient NOD-Rag1 null IL2rg null Ins2 Akita (NRG-Akita) mice and overcame progressively worsening hyperglycemia in these mice over several months . However, attempts to restore normal glycemia after transplantation of differentiated beta cells into immunodeficient animal models of diabetes have only shown a short-term amelioration at best, likely due to the rapid destruction of the transplanted beta cells [11, 15].
As an alternative possibility, nontumorigenic adult stem cells may be directly transplanted into animal models of T1DM to investigate their ability to differentiate in vivo into functional beta cells. Such an approach was recently investigated using bone marrow-derived mesenchymal stem cells  and umbilical cord-derived mesenchymal stem cells .
The life-long regenerative and remodeling capacities of skeletal muscle make it a potential niche for multipotent adult stems cells (reviewed in [22, 23]). Human skeletal muscle growth and regeneration can be triggered by muscle damage or increased activity and exercise, and involves activation of quiescent stem cells to proliferate and differentiate into de novo muscle fibers, connective tissue, vascularization, and peripheral neural cells [22, 24]. We have previously isolated, via serial pre-plating, a population of nonadherent muscle-derived stem cells (MDSC) that can differentiate into smooth, skeletal, and cardiac muscle lineages, as well as neuronal lineages . Although this multipotent differentiation implies an apparent heterogeneity of MDSC, like that of pluripotent ESC or iPSC, this heterogeneity is the signature of their multipotency as shown from similar adult muscle stem cells grown clonally  and revealing the expression of markers for the same multiple lineages as we described . Here, we examined the potential of multipotent adult stem cells isolated from skeletal muscle (MDSC) to differentiate towards another lineage—insulin-producing beta cells. This study reveals that MDSC not only have the capacity to spontaneously differentiate into insulin-expressing and insulin-secreting clusters of beta-like cells in vitro, but also can be used directly in vivo without predifferentiation by direct intraperitoneal (IP) injection into mouse models of T1DM where they are recruited to pancreatic islets within 48 h and differentiate into insulin-expressing beta-like cells within 10 days of injection. Finally, we show that, in mice with streptozotocin (STZ)-induced diabetes, hyperglycemic levels are reduced after injection of undifferentiated MDSC (an effect not seen in mice injected with saline alone). Considering their rapid purification from skeletal muscle and the absence of any predifferentiation step, MDSC offer a unique and promising approach for autologous beta-cell replacement therapies.
Cells extracted from skeletal muscle contain a nonadherent, nestin-enriched multipotent stem cell population—MDSC
MDSC differentiate in vitro into beta islet-like cell clusters expressing insulin
While data from immunohistochemistry, RT-PCR, and Western blot all show that the MDSC differentiated clusters expressed markers of immature and mature beta cells, it remained to be shown if they also secreted insulin in response to a glucose challenge. Several reports have demonstrated that different stem cells can be differentiated into mature insulin-secreting beta-cells in vitro [15–17], but only relatively few have shown that these cells effectively secrete insulin in a glucose-responsive manner [9, 20, 36, 37] (reviewed and discussed in [1, 6, 7, 38]). We therefore set up a standard insulin secretion assay using clusters differentiated in vitro for 8–10 weeks. For each measure, 4 to 5 islet-like cell clusters were collected in 24-well dishes and starved in KREBS buffer for 2 h before incubating for 1 h in KREBS supplemented with 2.8 mM glucose and subsequently incubated for 1 h in 20 mM glucose. Secreted insulin was measured by a FRET-based detection kit (CISBIO). Figure 4c shows that, when clusters were incubated for 1 h in 2.8 mM glucose, little or no insulin secretion was detected. In contrast, further stimulation for 1 h in 20 mM glucose resulted in a robust and consistent increase in insulin secretion (0.8–1 ng/ml of secreted insulin). Indeed, clusters secreted significant levels of insulin with regard to the small number of cells in the beta-like clusters (1500 to 2000 cells/well). Assays were performed in triplicate and similar results were obtained using at least three different preparations of MDSC-derived islet-like clusters, all showing effective insulin secretion in response to a 20 mM glucose challenge.
Taken together, these data show that both mouse and rat MDSC have the capacity to spontaneously differentiate in vitro into three-dimensional cell clusters that bear many of the hallmarks of mature pancreatic beta cells, and that beta islet-like cell clusters are competent to secrete insulin in a glucose-responsive manner.
In vivo homing to pancreatic islets and differentiation of MDSC into insulin-expressing beta cells in STZ-induced diabetic mice
These data show that donor stem cells were retrieved in the pancreatic islets of STZ-treated mice within 2 days and differentiated into insulin-expressing beta cells after 10–12 days. Therefore, in addition to their in vitro capacity to differentiate into islet-like cells, undifferentiated mouse MDSC can also differentiate in vivo into beta-like insulin-expressing cells within pancreatic islets of mice with STZ-induced beta-cell destruction.
Here we have shown that a population of multipotent progenitors isolated from skeletal MDSC are competent to differentiate in vitro into insulin-expressing and secreting islet-like cell clusters. These cell clusters express a number of typical markers of endocrine differentiation, pancreatic progenitor, and mature pancreatic beta-cell differentiation. The islet-like cell clusters differentiated in vitro expressed insulin as confirmed with fluorescent reporters (eGFP or mCherry) in clusters formed from MDSC extracted from MIP-eGFP or RIP-mCherry transgenic mouse muscles and also secreted insulin in response to a glucose challenge. Strikingly, when assayed in vivo, after MDSC freshly purified as stem cells were injected IP into mice with chemically induced beta-cell destruction, they specifically localized to damaged islets within 48 h and differentiated into insulin-expressing beta cells within 10 days of injection. In addition, injection of undifferentiated MDSC into hyperglycemic mice effectively reduced their blood glucose level towards normal levels.
Whereas mesenchymal stem cells derived from bone marrow and defined as a stromal cell population have been extensively investigated [13, 20, 21], the muscle stem cell population we have examined here was isolated and purified on the basis of its lower adherence to plastic and even collagen-coated dishes . Such low adherence properties may favor a higher migration capacity after systemic injection, and skeletal muscle displays a number of essential characteristics that make it a potentially ideal source of stem cells for direct use in vivo. With a life-long self-renewal capacity to repair damage or to undergo de novo growth and differentiation, skeletal muscle is remarkable by its paucity to develop cancer except in rare cases of rhabdomyosarcoma in childhood (reviewed in [40, 41]). This high plasticity of skeletal muscle, capable of rapidly undergoing atrophy or hypertrophy throughout life, involves the regeneration of different types of tissue and organs in addition to muscle fibers: connective tissue, vascularization, and, most importantly, innervation (since a denervated muscle cannot fully regenerate and instead undergoes atrophy ). The intermediate filament nestin, known to be a marker of meso-ectodermal stem and progenitor cells, is present in 12 to 15% of the cells initially isolated from muscle tissue and enriched fivefold (to 75–80%) during the MDSC purification process (Fig. 1c). Nestin is not only a marker of neuronal progenitor and stem cells  but is also expressed in progenitors cells present in pancreatic islets [8, 27, 28]. In addition, when pancreatic progenitors and insulin-expressing cells are derived from mouse ESC, nestin is expressed during the progenitor phase  and is shown to play essential roles in the process of stem cell differentiation into insulin-secreting cells . When insulin-expressing clusters were differentiated from MDSC expressing eGFP under the control of the nestin promoter, we observed that nestin-GFP-positive cells accumulated in and around islet-like clusters together with, but distinct from, insulin-expressing cells (Fig. 3a). We have previously shown that MDSC are multipotent and can differentiate into at least five different meso-ectodermal-derived lineages including typical neuronal-like cells  and automatically beating pacemaker cells (Mesirca et al., manuscript in preparation). Here we show that, in addition to these two excitable cell types, MDSC can also spontaneously differentiate within 2–3 weeks in culture into cell clusters that express several markers of another excitable cell type, the pancreatic beta cell lineage.
While several studies using pluripotent stem cells have shown their potential for differentiating into insulin-producing beta cells, including via ectodermal commitment [8, 11, 31, 45, 46] (reviewed in [1, 13, 14, 35]), few have shown a short-term ability of insulin-producing beta-like cells to decrease STZ-induced hyperglycemia in vivo when transplanted into encapsulated structures in immunodeficient mouse models of T1DM [9, 20] (reviewed in [13, 14]). Most studies have exploited relatively complex differentiation protocols lasting for 4–6 weeks and involving four or five distinct steps . In vitro, cultured MDSC spontaneously differentiated into insulin-expressing and -secreting beta-like cell clusters a few weeks after stem cell isolation and culture. These data add beta islet cells as a therapeutically important differentiation lineage to the expanding repertoire of cell types that can be derived from the MDSC multipotent muscle stem cell population .
Our principal objective, however, was to investigate the ability of MDSC to differentiate in vivo because undifferentiated stem cells retain greater plasticity, survival, and migration capacity. Indeed, there is growing evidence that mesenchymal stem cells which can be isolated from several adult tissues including bone marrow and adipose tissue (reviewed in [1, 6]) and neural crest-derived multipotent stem cells present in all peripherally innervated tissues (reviewed in ) may represent important cell sources for regenerative therapy. We have investigated here the in vivo behavior of undifferentiated MDSC injected into a mouse model of beta-cell deficiency induced by streptozotocin treatment. The nonteratogen character of MDSC (for up to 6 months follow-up after subcutaneous or IP injection into immunodeficient mice; VM, AF, and NJCL, unpublished observation) does not require their predifferentiation before use in vivo, and MDSC were transduced directly via IP or intravenous injection. We have examined their biodistribution within 48 h afterwards and found that MDSC are recruited to site(s) of injury, be it mechanical or chemically induced (Mitutsova et al., manuscript in preparation). Indeed undifferentiated purified MDSC (PP8) loaded with an in vivo cell tracer and directly transduced by IP injection were retrieved 48 h later in the beta cell-depleted pancreatic islets of STZ-treated mice and subsequently differentiated into insulin-expressing beta cells. The rapid islet-specific engraftment of MDSC after their IP injection brings support to an original report by Kozlova and Jansson showing that the migration and differentiation of neural crest stem cells are stimulated by pancreatic islets both in vitro and in vivo . We believe that this is the first example of specific homing of undifferentiated adult stem cells to damaged pancreatic islets and their subsequent differentiation into insulin-producing beta cells. In addition, MDSC were purified by a relatively fast and simple protocol using standard media and techniques, which are both positive aspects for a potential future therapeutic use. It remains to be determined if in vivo differentiated MDSC secrete sufficient insulin to durably restore fully normal glycemia levels in STZ-induced T1DM mouse models. Despite the high variability of the response to STZ amongst recipient mice , and known endogenous beta islet cell regeneration  and islet plasticity  in addition to the variable immunogenic response against MDSC after they differentiated into beta-like cells, our preliminary experiments in mice rendered hyperglycemic after a single high dose of STZ showed a partial reduction of the glycemia from 1 to 3 weeks after injection of MDSC (Fig. 7). Although encouraging, this result will require further confirmation to fully establish the insulin secretory phenotype and responses after engraftment of MDSC cells in beta cell-deficient animal models.
Overall, we show here that skeletal muscle-derived stem cells (MDSC) can undergo effective differentiation into insulin-expressing and -secreting beta-like cells both after in vitro culture and in vivo after systemic injection into mice with STZ-induced pancreatic beta cell destruction. The in vivo study represents, to our knowledge, the first demonstration of specific engraftment and differentiation of stem cells in pancreatic islets of diabetic mice. These data open a feasible therapeutic route through the potential use of MDSC by direct autologous injection of patient-derived stem cells as a promising alternative to the transplantation of differentiated and encapsulated beta cells.
Unless specified, all chemicals were obtained from Sigma-Aldrich, Saint-Quentin Fallavier, France. Cell tracker DiI (dioctadecyl-3-tetramethylindocarbocyanine perchlorate) was the chloro-methylbenzamido-DiI analog, CM-DiI, from Thermo-Fischer (Villebon-sur-Yvette, France) and used according to the manufacturer’s instructions.
Mice lines C57Bl6-wild type, NOD-MIP-eGFP (NOD/ShiLtJ-Tg(Ins1-EGFP/GH1); JAX-005282), and NOD-scid (NOD.CB17-Prkdc scid /J: JAX-001303) were from Jackson Labs (Bar Harbor, ME, USA), Black6-RIP-mCherry  and Nestin-GFP were from Dr. G. Enikolopov (Cold Spring Harbor Laboratory, NY, USA) . NOD-scid-MIP-eGFP mice were obtained by crossing NOD-scid and NOD-MIP-eGFP. Sprague Dawley rats were from Charles River laboratory, and all animals were maintained in the IGH/IGF transgenic mouse facility. All animal handling strictly conformed to university, local, and international animal husbandry, veterinary care, and ethics rules (see below).
Gerbil fibroma fibroblasts (CCL-146) were maintained in DME supplemented with 10% v/v serum as described previously . MIN6 cells were cultured in DMEM (Gibco-LifeSciences, ThermoFischer, Villebon-sur-Yvette, France) supplemented with 25 mM glucose, 16% fetal bovine serum (FBS; Pall-Biosepra, Cergy-Saint-Christophe, France), penicillin/streptomycin (Gibco), and 55 μM β-mercaptoethanol (BME).
Unless stated otherwise, antibodies were obtained from the Developmental Studies Hybridoma Bank (DSHB, Iowa, USA); for IHC and Western blotting: anti-eGFP chicken polyclonal GFP-1020 (Aves Inc, Tigard, OR, USA; 1/500); mouse monoclonal against: insulin (I2018; Sigma; 1/1000), Nestin (Rat-401; 1/200), PDX1 (F109-D12; 1/500), Nkx6.1 (F55-A10; 1/200), Nkx2.2 (74.5A5; 1/200), Pax6 (PAX6; 1/500); rabbit polyclonal antibodies against: MafA (A300-611A; Bethyl-Laboratories, Montgomery, Texas, USA) and Glut2 (H-67; Santa-Cruz Biotechnologies, CliniSciences, Nanterre, France).
Isolation and purification of MDSC
After surgical removal, hind leg muscles were subject to enzymatic dissociation (0.2% (w/v) collagenase A (Roche, Basel, Switzerland) and 1 mg/ml dispase (Gibco) for 45 min), the crude muscle cell-containing fraction was isolated by filtration and centrifugation essentially as described before . Subsequently, after a short 40-min incubation to allow fibroblasts to adhere, the MDSC-containing supernatant was recovered and MDSC collected by centrifugation (576 × g, 10 min) at room temperature (RT). Pellets were resuspended in proliferation media (PM; DME/Hams F12 mixture, supplemented with 16% (v/v) fetal calf serum (FCS; Gibco), 1% Ultroser G (Pall-BioSepra), 2 mM l-glutamine, antibiotic/antimycotic mix (Gibco), and 100 μM BME (added freshly from a 100 mM stock in phosphate-buffered saline (PBS)) and plated at a density of 2.5 million cells/100-mm dish as described previously . After 24 h, unattached cells were gently detached by pipetting and collected by centrifugation (576 × g, 10 min). Pelleted cells were resuspended in 10 ml fresh media and transferred to a new 100-mm dish. MDSC were purified by eight rounds of this plating technique performed every 24 h which ensures that the nonadherent MDSC fraction is always passed daily into fresh growth factor-rich media. Figure 1a shows a schematic overview of the pre-plating process and typical examples of the different cell phenotypes that arise after MDSC from PP8 are allowed to adhere (on adherent cells from previous pre-plates, inactivated CCL-146 fibroblasts, or laminin-coated dishes ) and differentiate for 1 to 3 weeks. Exactly the same protocol was followed for producing rat MDSC.
For RT-PCR analysis of stem cells, MDSC were obtained after seven serial pre-plates (PP8). For differentiated cell clusters, MDSC (PP8) were seeded on matrix-providing adherent cells of earlier pre-plates or inactivated fibroblasts (CCL-146) and islet-like clusters formed 15–20 days thereafter. Clusters were picked from adherent cell monolayers (8 to 12 clusters 6 weeks after plating) and total RNA was extracted using RNeasy (Quiagen, Germany). cDNA was generated using SuperScript III Reverse Transcriptase (Invitrogen, ThermoFischer, Villebon-sur-Yvette, France) according to the manufacturer’s protocol using 50 nM oligo(dT)20. PCR was performed for 35 cycles at an initial denaturing temperature of 94 °C for 3 min, denaturing temperature of 94 °C for 45 s, annealing temperature of 55 °C for 30 s, extension temperature of 72 °C for 90 s, and final extension at 72 °C for 10 min using GoTaq polymerase (Promega, Charbonnières-les-Bains, France).
Western blot analysis
For protein analysis, proteins were isolated, separated by SDS-PAGE, and analyzed by Western blot on PVDF membranes as described previously . Briefly, nonadherent cells (PP8) were washed once in ice-cold PBS and collected by centrifugation at 570 × g for 10 min before freezing as pellets until use. Adherent differentiated cells, including clusters, were washed once in ice-cold PBS and, after manual detachment, collected by centrifugation (570 × g for 10 min) before freezing as pellets until use. Frozen pellets were resuspended in 40 mM Tris-Cl pH 6.8, 1.0 mM EDTA, 1% (w/v) SDS, and 7.5% (v/v) glycerol, and cell DNA was sheared by 10 passages through a 26G needle, cleared by brief centrifugation, and the protein concentration of the supernatant determined by Lowry assay (BioRad, Mitry-Mory, France). Subsequently, samples were diluted with an equal volume of 200 mM Tris-Cl pH 6.8, 4.0 mM EDTA, 4% (w/v) SDS, 30% (v/v) glycerol, 400 mM DTT, and 0.03% (w/v) bromophenol blue. Ten to 15 μg total protein/lane were resolved on 12.5 or 10% SDS-PAGE gels and transferred to PVDF-immobilon (Millipore, Merck, Fontenay sous Bois, France). Following transfer, membranes were stained with amido-black to confirm loading homogeneity before blocking by incubation in 1% (w/v) PVP for 15 min followed by 30 min incubation in TBS containing 0.5% (w/v) BSA and 0.5% (w/v) skimmed milk. Membranes were incubated with primary antibodies overnight at 4 °C in the same buffer.
Insulin secretion was assayed by FRET-based assay using an insulin homogeneous time resolved fluorescence (HTRF) kit (Cisbio International, Codolet, France) according to the manufacturer’s instructions. Twenty-four hours before the assay, islet-like clusters were transferred to low-glucose DME (5.6 mM, 1 g/l). The next day, islet-like cell clusters (4–5 clusters) were transferred to 24-well plates and washed 3× with 1 ml Hanks balanced salt solution and then starved in 250 μl sterile filtered Krebs-Ringer solution (KR; 119 mM NaCl, 4 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 20 mM HEPES, 5 mM NaHCO3 pH 7.5, and 0.1% (w/v) BSA Fraction 5 ultra-pure (Sigma-Aldrich)) for 2 h. After starvation, the KR solution was collected (T0) and replaced by 250 μl KR supplemented with 2.8 mM glucose, and clusters were incubated for 1 h. After collection of the ringer solution (T2.8), cells were washed 3× with KR before transfer to KR supplemented with 20 mM glucose and further incubated for 1 h (T20). Culture supernatants were stored frozen at –20 °C until analysis. Insulin released during incubation was quantified in triplicate and for at least three separate batches of clusters.
For immunofluorescence analysis of whole clusters in situ, dishes were fixed in 3.7% (v/v) formalin in PBS for 10 min at RT, extracted with 0.1% triton X100 in PBS for 30 s, and then incubated in 0.5% (w/v) BSA in PBS (PBS-BSA) for 15 min to block unspecific binding. Areas surrounding clustered cells were demarked on the dish, and clusters were incubated with primary and secondary antibodies diluted in PBS-BSA essentially as described before . Alternatively, clusters (approximately 10–15) were mechanically harvested from adherent monolayers and enzymatically dissociated by brief incubation in 0.1% (w/v) collagenase and 1 mg/ml dispase for 2 min at RT. After enzyme inactivation by the addition of PBS-BSA supplemented with 250 μM EDTA, cells were collected by cytospin (Thermo-Fisher Scientific, Paisley, UK), and cells were fixed and processed for immunofluorescence as described previously .
The islet-like cell clusters were stained with dithizone (DTZ; Sigma-Aldrich) solution to identify the zinc-enriched insulin-expressing cells . Clusters were incubated in 0.005% (w/v) DTZ in filter sterilized (0.45 μm) culture media for 15 min at 37 °C, and washed three times with warm PBS before direct microscopic examination for insulin-producing cells, which are selectively stained Crimson red.
In vivo assay of MDSC differentiation in STZ-treated mice
Chemically induced beta-cell destruction was performed by injection of the glucose homologue streptozotocin (STZ) using the high-dose strategy (reviewed in ). NOD-scid-MIP-eGFP mice were fasted for 6 h prior to STZ treatment by intraperitoneal (IP) injection of a single dose of STZ at 150 mg/kg (freshly prepared in 100 mM Na-citrate pH 4.5 and used within 15 min). Mice were transplanted 1 h afterward via IP injection of 2–3 × 105 MDSC stem cells in 0.2 ml saline buffer isolated from RIP-mCherry mouse muscles, or mock transplanted with IP injection of saline buffer.
Immunofluorescence analysis on mouse pancreas
Pancreas were harvested and rinsed with PBS prior to fixation with 4% paraformaldehyde (PFA) at 4 °C for 1 h. The PFA was discarded and the pancreas washed in PBS and then kept overnight at 4 °C in 30% sucrose. For the preparation of vibratome tissue slides, the pancreas was minced into small pieces of about 0.4 mm2 and placed in a plastic mold. Agarose gel (4%) was cast on the tissues to provide firm holding for 150-μm sectioning (Vibratome Leica VT1000 S, Wetzlar, Germany). Alternatively, pancreas tissue fixed and equilibrated in 30% sucrose was embedded in OCT before cryosectioning (10-μm thick sections). The sections were incubated with 10% goat serum (Abcam, Cambridge, MA, USA), diluted in 0.1% Triton X-100/0.5% BSA/PBS for 1 h at room temperature, and then washed in PBS before incubation with primary antibody for 48 h at 4 °C. Subsequent incubation with secondary antibody along with Hoechst 33358 for nuclear staining was for 2 h at room temperature. Both the primary and secondary antibodies were diluted in 0.1% Triton X-100/0.5% BSA/PBS. After rinsing with PBS, sections were incubated for 5 min with 1% Sudan Black B in 70% ethanol and then washed with 70% ethanol for 5 min and distilled water for 5 min at room temperature to reduce autofluorescence.
Blood glucose measurements in mice treated with STZ
Blood glucose levels were measured at the tail tip of 6-month-old C57Bl6 wild-type males (n = 8) at the same time (2 pm; corresponding to the middle of the inactive resting day time) every 3 to 4 days before and after a single IP injection of 150 mg/kg STZ in citrate buffer. When the blood glucose levels reached hyperglycemic values above 450 mg/dl, half of the mice (n = 4) were injected IP with 300,000 MDSC purified from the muscle of wild-type female co-sanguineous mice and glycemic levels were compared over 4 weeks in the two groups of mice.
Enhanced green fluorescent protein
embryonic stem cells
Induced pluripotent stem cells
Muscle-derived stem cells
Mouse insulinoma cell line 6
Mouse Ins-1 insulin promoter
Rat-INS2 insulin promoter
Type I diabetes mellitus
We thank Dr. G Enikolopov (Cold Spring Harbor Laboratory, NY, USA) for providing the transgenic mouse line Nestin-eGFP described in . The hybridoma monoclonal antibodies Nestin (Rat-401) developed by S. Hockfield, PDX1 (F109-D12) and Nkx6.1 (F55-A10) developed by OD Madsen, Nkx2.2 (74.5A5) developed by TM Jessell and S. Brenner-Morton, and Pax6 (PAX6) developed by A. Kawakami were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242, USA.
Funding for this project was provided by the Association pour la Recherche sur le Diabète (ARD, France; http://www.a-rd.fr) and the Fondation pour la Recherche Medicale (FRM; ref: DPC20111122986, https://www.frm.org) to AF; Fundamental Research Grant Scheme (FRGS) (Ref: FRGS/1/2014/SKK01/UPM/02/1) to SA; VM was supported by the l'Association Française contre les Myopathies (AFM; http://www.afm-telethon.com) and WWYY was supported by an Erasmus Mundus (MAHEVA program; http://www.maheva.eu) scholarship.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
AF and NJCL: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript, and financial support; VM and WWYY: collection and assembly of data, data analysis and interpretation, conception and design, and final approval of manuscript; RD and CF: collection and assembly of data, and final approval of manuscript; MS: development of mouse models, data analysis and interpretation, and final approval of manuscript; EHH, SA, and PM: data analysis and interpretation, and final approval of manuscript.
The authors declare that they have no competing interests.
This study conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and to European directives (2010/63/EU). All animal studies were approved by the French Ministry of Agriculture and the Languedoc Roussillon Institutional Animal Care, Experimental Ethics and Use Committee (CEEA-LR-1053). Agreement N° C-34-172-13.
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