Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral “rejuvenating” factors that can restore regenerative function. Here, we demonstrate that the circulating protein Growth Differentiation Factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction.
Aging tissues experience a progressive decline in homeostatic and regenerative capacities, which has been attributed to degenerative changes in tissue-specific stem cells, stem cell niches and systemic cues that regulate stem cell activity. Understanding the molecular pathways involved in this age-dependent deterioration of stem cell function will be critical for developing new therapies for diseases of aging that target the specific causes of age-related functional decline. Here we explore key molecular pathways that are commonly perturbed as tissues and stem cells age and degenerate. We further consider experimental evidence both supporting and refuting the notion that modulation of these pathways per se can reverse aging phenotypes. Finally, we ask whether stem cell aging establishes an epigenetic ‘memory’ that is indelibly written or one that can be reset.
The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGFβ superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a new therapeutic opportunity for cardiac aging.
Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal three-dimensional (3D) co-cultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell co-cultures. Gain and loss of function experiments showed BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of Thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1-null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide new tools to understand the mechanisms of respiratory diseases at the single cell level.
Lung stem cells; Lineage; Differentiation; Endothelial cells; Tsp1; Bmp4
Since the advent of tyrosine kinase inhibitors as targeted therapies in cancer, several receptor tyrosine kinases (RTKs) have been identified that are operationally important for disease progression. Rhabdomyosarcoma (RMS) is an example of a malignancy in need of new treatment options, and a better understanding of the heterogeneity of RTK expression could advance this goal. We investigated populations of alveolar RMS (aRMS) tumor cells derived from a transgenic mouse model expressing two such RTKs, Pdgfrα and Igf1r, by fluorescent activated cell sorting. Sorted subpopulations that were positive or negative for Pdgfrα and Igf1r dynamically altered their RTK cell surface expression profile as early as the first cell division. Interestingly, too, a difference in total Pdgfrα expression and nuclear Igf1r expression was conserved in populations. Nuclear Igf1r expression was greater than cytoplasmic Igf1r in cells with initially high cell surface Igf1r. Cells with high nuclear expression of Igf1r established tumors most efficiently in vivo. RNAi-mediated silencing of Igf1r in the subpopulation of cells initially harboring higher cell surface and total Igf1r resulted in a comparatively greater reduction in anchorage-independent colony formation than sorted with initially lower cell surface and total Igf1r expression. In accordance with our findings in murine aRMS, human aRMS were also found to express nuclear Igf1r.
Pdgfrα; Igf1r; rhabdomyosarcoma; nucleus; receptor tyrosine kinase
Patients with type 1 diabetes (T1D) suffer excessive morbidity and mortality following myocardial infarction (MI) that is not fully explained by the metabolic effects of diabetes. Acute MI is known to trigger a profound innate inflammatory response with influx of mononuclear cells and production of proinflammatory cytokines that are crucial for cardiac repair. We hypothesized that these same pathways might exert ‘adjuvant effects’ and induce pathological responses in autoimmune-prone T1D hosts. Here we show that experimental MI in nonobese diabetic (NOD) mice - but not in control C57BL/6 mice - results in a severe post-infarction autoimmune (PIA) syndrome characterized by destructive lymphocytic infiltrates in the myocardium, infarct expansion, sustained cardiac IgG autoantibody production and Th1 effector cell responses against cardiac (α-)myosin. PIA was prevented by inducing tolerance to α-myosin, demonstrating that immune responses to cardiac myosin are required for this disease process. Extending these findings to humans, we developed a panel of immunoassays for cardiac autoantibody detection and found autoantibody positivity in 83% post-MI T1D patients. We further identified shared cardiac myosin autoantibody signatures between post-MI T1D patients and non-diabetic patients with myocarditis – that were absent in post-MI type 2 diabetic patients - and confirmed the presence of myocarditis in T1D by cardiac magnetic resonance imaging techniques. These data provide experimental and clinical evidence for a distinct post-MI autoimmune syndrome in T1D. Our findings suggest that PIA may contribute to worsened post-MI outcomes in T1D, and highlight a role for antigen-specific immunointervention to selectively block this pathway.
Long recognized to be potent suppressors of immune responses, Foxp3+CD4+ regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloidlineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.
The landmark discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka has transformed regenerative biology. Previously, insights into the pathogenesis of chronic human diseases have been hindered by the inaccessibility of patient samples. However, scientists are now able to convert patient fibroblasts into iPSCs and differentiate them into disease-relevant cell types. This ability opens new avenues for investigating disease pathogenesis and designing novel treatments. In this review, we highlight the uses of human iPSCs to uncover the underlying causes and pathological consequences of diabetes and metabolic syndromes, multi-factorial diseases whose etiologies have been difficult to unravel using traditional methodologies.
Multipotent stromal cells (MSC) and their osteoblastic lineage cell (OBC) derivatives are part of the BM niche and contribute to hematopoietic stem cells (HSC) maintenance. Here, we show that myeloproliferative neoplasia (MPN) progressively remodels the endosteal BM niche into a self-reinforcing leukemic niche that impairs normal hematopoiesis, favors leukemic stem cell (LSC) function and contributes to BM fibrosis. We show that leukemic myeloid cells stimulate MSCs to overproduce functionally altered OBCs, which accumulate in the BM cavity as inflammatory myelofibrotic cells. We identify roles for TPO, CCL3 and direct cell-cell interactions in driving OBC expansion, and for changes in TGFβ, Notch and inflammatory signaling in OBC remodeling. MPN-expanded OBCs, in turn, exhibit decreased expression of many HSC retention factors and severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue to treat MPN patients and prevent myelofibrosis.
Calorie restriction (CR) extends lifespan and ameliorates age-related pathologies in most species studied; yet the mechanisms underlying these effects remain unclear. Using mouse skeletal muscle as a model, we show that CR acts in part by enhancing the function of tissue-specific stem cells. Even short-term CR significantly enhanced stem cell availability and activity in the muscle of young and old animals, in concert with an increase in mitochondrial abundance and induction of conserved metabolic and longevity regulators. Moreover, CR enhanced endogenous muscle repair and CR initiated in either donor or recipient animals improved the contribution of donor cells to regenerating muscle following transplant. These studies indicate that metabolic factors play a critical role in regulating stem cell function and that this regulation can influence the efficacy of recovery from injury and the engraftment of transplanted cells.
Selectins are carbohydrate-binding adhesion molecules critically involved in leukocyte recognition of endothelium. The endothelial selectins have been implicated in homing of hematopoietic stem/progenitor cell(s) (HSPC) to the bone marrow (BM) during bone marrow transplant (BMT), but the precise role(s) of individual selectins in this process have never been defined. BMT of lethally irradiated mice lacking both endothelial selectins (E/P KO) with limiting numbers of wild-type BM cells rescued significantly fewer E/P KO than WT recipients, but higher numbers of transplanted WT cells rescued E/P KOs in a dose-dependent fashion. Short term homing assays confirmed a substantial defect in HSPC homing to BM in E/P KO mice. In contrast, BMT of E-selectin null (E KO) or P-selectin null (P KO) mice at limiting cell number uniformly rescued >95% of the transplanted animals. Consistent with these functional results, flow cytometric analysis revealed both E-selectin ligands and P-selectin ligands on distinct subsets of HSPC. Taken together, these results demonstrate overlapping functions for the endothelial selectins in HSPC homing to BM in the setting of BMT, and define a novel aspect of HSPC heterogeneity linked to selectin ligand expression.
hematopoietic stem cell; selectin; bone marrow transplantation; homing
In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) resulted in the expansion and directed differentiation of endogenous heart progenitors in a murine myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors, and temporal control with VEGF inhibitors, documented the markedly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long term organ repair.
Haematopoietic stem cells (HSCs) are multipotent, self-renewing progenitors that generate all mature blood cells. HSC function is tightly controlled to maintain haematopoietic homeostasis, and this regulation relies on specialized cells and factors that constitute the haematopoietic ‘niche’, or microenvironment. Recent discoveries, aided in part by technological advances in in vivo imaging, have engendered a new appreciation for the dynamic nature of the niche, identifying novel cellular and acellular niche components and uncovering fluctuations in the relative importance of these components over time. These new insights significantly improve our understanding of haematopoiesis and raise fundamental questions about what truly constitutes a stem cell niche.
Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.
The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia/macrophages, which can be polarized to distinct functional phenotypes: proinflammatory (M1) or anti-inflammatory/immunoregulatory (M2). Here we show that a switch from an M1- to M2-dominant response occurred within microglia and peripherally-derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 conditioned media, and impaired in vivo following intra-lesional M2 depletion. M2 densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger animal, and in MS lesions that normally show remyelination. Blocking M2-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Our results therefore show that M2 polarization is essential for efficient remyelination and identify activin-A as a novel therapeutic target for CNS regeneration.
Ligand-independent, constitutive activation of Hedgehog signalling in mice expressing a mutant, activated SmoM2 allele results in the development of multifocal, highly differentiated tumours that express myogenic markers (including desmin, actin, MyoD and myogenin). The histopathology of these tumours, commonly classified as rhabdomyosarcomas, more closely resembles human fetal rhabdomyoma (FRM), a benign tumour that can be difficult to distinguish from highly differentiated rhabdomyosarcomas. We evaluated the spectrum of Hedgehog (HH) pathway gene mutations in a cohort of human FRM tumours by targeted Illumina sequencing and fluorescence in situ hybridization testing for PTCH1. Our studies identified functionally relevant aberrations at the PTCH1 locus in three of five FRM tumours surveyed, including a PTCH1 frameshift mutation in one tumour and homozygous deletions of PTCH1 in two tumours. These data suggest that activated Hedgehog signalling contributes to the biology of human FRM.
fetal rhabdomyoma; hedgehog signalling; PTCH1
Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.
Rare childhood cancers can be paradigms from which important new principles can be discerned. The childhood muscle cancer rhabdomyosarcoma is no exception, having been the focus of the original 1969 description by Drs. Li and Fraumeni of a syndrome now know to be commonly caused by underlying p53 tumor suppressor loss-of-function. In our studies using a conditional genetic mouse model of alveolar rhabdomyosarcoma in conjunction with human tumor cell lines, we have uncovered that the expression level of a translocation-mediated fusion gene, Pax3:Foxo1a, is dynamic and varies during the cell cycle. Our studies support that Pax3:Foxo1a facilitate the yeast-related process of checkpoint adaptation under stresses such as irradiation. The broader implication of our studies is that distal cis elements (promoter-influencing regions of DNA) may be critical to fully understanding the function of cancer-associated translocations.
Fluorescence-activated cell sorting (FACS) strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA) cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells. These CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45−CD11b−GlyA−CD31−CD34+ fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7+ CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis.
•CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi human fetal muscle cells are PAX7+•CD34−CD56intITGA7hi fetal cells have bipotent myogenic/osteogenic activity in vitro•PAX7+ CD34−CD56intITGA7hi cells are fewer in number in adult versus fetal muscle
This work by Castiglioni et al. establishes the presence of functionally distinct cell populations within the pool of myofiber-associated cells in human fetal and adult skeletal muscle and reports effective methods for the direct isolation by fluorescence-activated cell sorting of a cell population in fetal muscle with bipotent in vitro myogenic/osteogenic activity.
Mutations in the Survival of Motor Neuron (SMN) gene underlie the development of spinal muscular atrophy (SMA), which currently represents the leading genetic cause of mortality in infants and toddlers. SMA is characterized by degeneration of spinal cord motor neurons and muscle atrophy. Although SMA is often considered to be a motor neuron disease, accumulating evidence suggests that muscle cells themselves may be affected by low levels of SMN. Here, we examine satellite cells, tissue-resident stem cells that play an essential role in the growth and repair of skeletal muscle, isolated from a severe SMA mouse model (Smn−/−; SMN2+/+). We found similar numbers of satellite cells in the muscles of SMA and wild-type (Smn+/+; SMN2+/+) mice at postnatal day 2 (P2), and, when isolated from skeletal muscle using cell surface marker expression, these cells showed comparable survival and proliferative potential. However, SMA satellite cells differentiate abnormally, revealed by the premature expression of muscle differentiation markers, and, especially, by a reduced efficiency in forming myotubes. These phenotypes suggest a critical role of SMN protein in the intrinsic regulation of muscle differentiation and suggest that abnormal muscle development contributes to the manifestation of SMA symptoms.
Spinal muscular atrophy; Satellite cells; Muscle
Hematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs.
•HSCs are transcriptionally primed for rapid activation•A total of 322 HSC-enriched genes, including 51 transcription factors, were identified•Ectopic Hlf expression increased ex vivo self-renewal in stem and progenitor cells•Analysis revealed a transcription factor family hypothesized to regulate multipotency
Remyelination is a regenerative process in the central nervous system (CNS) that produces new myelin sheaths from adult stem cells. The decline in remyelination that occurs with advancing age poses a significant barrier to therapy in the CNS, particularly for long-term demyelinating diseases such as multiple sclerosis (MS). Here we show that remyelination of experimentally-induced demyelination is enhanced in old mice exposed to a youthful systemic milieu through heterochronic parabiosis. Restored remyelination in old animals involves recruitment to the repairing lesions of blood-derived monocytes from the young parabiotic partner, and preventing this recruitment partially inhibits rejuvenation of remyelination. These data suggest that enhanced remyelinating activity requires both youthful monocytes and other factors, and that remyelination-enhancing therapies targeting endogenous cells can be effective throughout life.
Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively-isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.
Integrative organ crosstalk regulates key aspects of energy homeostasis, and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that crosstalk between the liver and pancreatic islets modulates β cell growth in response to insulin resistance, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary in vivo parabiosis and transplantation assays, as well as in vitro islet culture approaches, we demonstrate that humoral, nonneural, non-cell-autonomous factor(s) induces β cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human β cell proliferation in ex vivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of β cell growth factor(s) in insulin-resistant states.
Several studies have reported that bone marrow (BM) cells may give rise to neurons and astrocytes in vitro and in vivo. To further test this hypothesis, we analyzed for incorporation of neural cell types expressing donor markers in normal or injured brains of irradiated mice reconstituted with whole BM or single, purified c-kit+Thy1.1loLin−Sca-1+ (KTLS) hematopoietic stem cells (HSCs), and of unirradiated parabionts with surgically anastomosed vasculature. Each model showed low-level parenchymal engraftment of donor-marker+ cells with 96–100% immunoreactivity for panhematopoietic (CD45) or microglial (Iba1 or Mac1) lineage markers in all cases studied. Other than one arborizing structure in the olfactory bulb of one BM-transplanted animal, possibly representing a neuronal or glial cell process, we found no donor-marker–expressing astrocytes or non-Purkinje neurons among >10,000 donor-marker+ cells from 21 animals. These data strongly suggest that HSCs and their progeny maintain lineage fidelity in the brain and do not adopt neural cell fates with any measurable frequency.