Skeletal muscle (SkM) regeneration relies on the activity of myogenic progenitors that reside beneath the basal lamina of myofibers. Here, we describe a protocol for the isolation of the SkM ...progenitors from young and old mice by exploiting their outgrowth potential from SkM explants on matrigel coated dishes in the presence of high serum, chicken embryo extract and basic fibroblast growth factor. Compared to other protocols, this method yields a higher number of myoblasts (10–20 million) by enabling the outgrowth of these cells from tissue fragments. The majority of outgrowth cells (~90%) were positive for myogenic markers such as α7-integrin, MyoD, and Desmin. The myogenic cell population could be purified to 98% with one round of pre-plating on collagen coated dishes, where differential attachment of fibroblasts and other non-myogenic progenitors separates them from myoblasts. Moreover, the combination of high serum medium and matrigel coating provided a proliferation advantage to myogenic cells, which expanded rapidly (~24 h population doubling), while non-myogenic cells diminished over time, thereby eliminating the need for further purification steps such as FACS sorting. Finally, myogenic progenitors gave rise to multinucleated myotubes that exhibited sarcomeres and spontaneous beating in the culture dish.
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•Efficient method for isolation of myogenic progenitors from skeletal muscle explants•High yield of isolation (10–20 million myoblasts from the hindlimb muscles of one mouse)•Outgrowth cells are ~90% myoblasts and the purity increases up to 98% with one round of pre-plating.•The isolated myoblasts can differentiate into multinucleated myofibers capable of spontaneous contraction.
Intercellular adhesion through homotypic interaction between cadherins regulates multiple cellular processes including cytoskeletal organization, proliferation, and survival. In this paper, we ...provide evidence that cadherin‐11 (CDH11) binds to and promotes cell proliferation both in vitro and in vivo in synergy with the platelet‐derived growth factor receptor beta (PDGFRβ). Engagement of CDH11 increased the sensitivity of cells to PDGF‐BB by 10‐ to 100‐fold, resulting in rapid and sustained phosphorylation of AKT, ultimately promoting and cell proliferation and tissue regeneration. Indeed, wound healing experiments showed that healing was severely compromised in Cdh11−/− mice, as evidenced by significantly decreased proliferation, AKT phosphorylation, and extracellular matrix (ECM) synthesis of dermal cells. Our results shed light into understanding how intercellular adhesion can promote cell proliferation and may have implications for tissue regeneration and cancer progression.
Our laboratory reported the derivation of neural crest stem cell (NCSC)-like cells from the interfollicular epidermis of the neonatal and adult epidermis. These keratinocyte (KC)-derived Neural Crest ...(NC)-like cells (KC-NC) could differentiate into functional neurons, Schwann cells (SC), melanocytes, and smooth muscle cells in vitro. Most notably, KC-NC migrated along stereotypical pathways and gave rise to multiple NC derivatives upon transplantation into chicken embryos, corroborating their NC phenotype. Here, we present an innovative design concept for developing anisotropically aligned scaffolds with chemically immobilized biological cues to promote differentiation of the KC-NC towards the SC. Specifically, we designed electrospun nanofibers and examined the effect of bioactive cues in guiding KC-NC differentiation into SC. KC-NC attached to nanofibers and adopted a spindle-like morphology, similar to the native extracellular matrix (ECM) microarchitecture of the peripheral nerves. Immobilization of biological cues, especially Neuregulin1 (NRG1) promoted the differentiation of KC-NC into the SC lineage. This study suggests that poly-ε-caprolactone (PCL) nanofibers decorated with topographical and cell-instructive cues may be a potential platform for enhancing KC-NC differentiation toward SC.
Although soluble factors, such as transforming growth factor β1 (TGF-β1), induce mesenchymal stem cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage, the role of adherens ...junctions in this process is not well understood. In this study, we found that cadherin-11 but not cadherin-2 was necessary for MSC differentiation into SMCs. Cadherin-11 regulated the expression of TGF-β1 and affected SMC differentiation through a pathway that was dependent on TGF-β receptor II (TGFβRII) but independent of SMAD2 or SMAD3. In addition, cadherin-11 activated the expression of serum response factor (SRF) and SMC proteins through the Rho-associated protein kinase (ROCK) pathway. Engagement of cadherin-11 increased its own expression through SRF, indicative of the presence of an autoregulatory feedback loop that committed MSCs to the SMC fate. Notably, SMC-containing tissues (such as aorta and bladder) from cadherin-11-null (Cdh11(-/-)) mice showed significantly reduced levels of SMC proteins and exhibited diminished contractility compared with controls. This is the first report implicating cadherin-11 in SMC differentiation and contractile function in vitro as well as in vivo.
Hyposalivation reduces the patient quality of life, as saliva is important for maintaining oral health. Current treatments for hyposalivation are limited to medications such as the muscarinic ...receptor agonists, pilocarpine and cevimeline. However, these therapies only provide temporary relief. Therefore, alternative therapies are essential to restore salivary gland function. An option is to use bioengineered scaffolds to promote functional salivary gland regeneration. Previous studies demonstrated that the laminin-111 protein is critical for intact salivary gland cell cluster formation and organization. However, laminin-111 protein as a whole is not suitable for clinical applications as some protein domains may contribute to unwanted side effects such as degradation, tumorigenesis and immune responses. Conversely, the use of synthetic laminin-111 peptides makes it possible to minimize the immune reactivity or pathogen transfer. In addition, it is relatively simple and inexpensive as compared to animal-derived proteins. Therefore, the goal of this study was to demonstrate whether a 20 day treatment with laminin-111-derived peptide conjugated fibrin hydrogel promotes tissue regeneration in submandibular glands of a wound healing mouse model. In this study, laminin-111-derived peptide conjugated fibrin hydrogel significantly accelerated formation of salivary gland tissue. The regenerated gland tissues displayed not only structural but also functional restoration.
A major limitation in engineering vascular grafts is the lack of proper endothelium to prevent thrombosis and subsequent graft failure. Obtaining endothelial cells from patients' vasculature is ...intrusive and requires extensive culture time. Here we present an alternative strategy wherein abundant and easily accessible monocytes from peripheral blood are cultured and differentiated towards an endothelial-like state capable of preventing thrombosis through production of nitric oxide and formation of endothelial adherens junctions. Considering the plethora of monocytes present within peripheral blood, this method provides a robust alternative to generating endothelial cells required for vascular graft production.
Although the therapeutic potential of mesenchymal stem cells (MSCs) is widely accepted, loss of cell function due to donor aging or culture senescence are major limiting factors hampering their ...clinical application. Our laboratory recently showed that MSCs originating from older donors suffer from limited proliferative capacity and significantly reduced myogenic differentiation potential. This is a major concern, as the patients most likely to suffer from cardiovascular disease are elderly. Here we tested the hypothesis that a single pluripotency‐associated transcription factor, namely Nanog, may reverse the proliferation and differentiation potential of bone marrow‐derived MSC (BM‐MSC) from adult donors. Microarray analysis showed that adult (a)BM‐MSC expressing Nanog clustered close to Nanog‐expressing neonatal cells. Nanog markedly upregulated genes involved in cell cycle, DNA replication, and DNA damage repair and enhanced the proliferation rate and clonogenic capacity of aBM‐MSC. Notably, Nanog reversed the myogenic differentiation potential and restored the contractile function of aBM‐MSC to a similar level as that of neonatal (n)BM‐MSC. The effect of Nanog on contractility was mediated—at least in part—through activation of the TGF‐β pathway by diffusible factors secreted in the conditioned medium of Nanog‐expressing BM‐MSC. Overall, our results suggest that Nanog may be used to overcome the effects of organismal aging on aBM‐MSC, thereby increasing the potential of MSC from aged donors for cellular therapy and tissue regeneration. STEM CELLS 2012;30:2746–2759
We investigate the age-related metabolic changes that occur in aged and rejuvenated myoblasts using in vitro and in vivo models of aging. Metabolic and signaling experiments reveal that human ...senescent myoblasts and myoblasts from a mouse model of premature aging suffer from impaired glycolysis, insulin resistance, and generate Adenosine triphosphate by catabolizing methionine via a methionine adenosyl-transferase 2A-dependant mechanism, producing significant levels of ammonium that may further contribute to cellular senescence. Expression of the pluripotency factor NANOG downregulates methionine adenosyltransferase 2 A, decreases ammonium, restores insulin sensitivity, increases glucose uptake, and enhances muscle regeneration post-injury. Similarly, selective inhibition of methionine adenosyltransferase 2 A activates Akt2 signaling, repairs pyruvate kinase, restores glycolysis, and enhances regeneration, which leads to significant enhancement of muscle strength in a mouse model of premature aging. Collectively, our investigation indicates that inhibiting methionine metabolism may restore age-associated impairments with significant gain in muscle function.
Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to ...its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain from Laminin-111 and the YIGSR peptide corresponding to the β1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens.
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