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In JoVE (1)
Other Publications (14)
- Journal of Cellular Biochemistry. Supplement
- The Journal of Clinical Investigation
- Experimental Hematology
- Stem Cells (Dayton, Ohio)
- The Journal of Biological Chemistry
- The Journal of Clinical Investigation
- Cell Stem Cell
- Proceedings of the National Academy of Sciences of the United States of America
- Stem Cells (Dayton, Ohio)
- Stem Cells (Dayton, Ohio)
- Stem Cells (Dayton, Ohio)
- Journal of Cardiovascular Pharmacology
- Stem Cells (Dayton, Ohio)
- American Journal of Physiology. Lung Cellular and Molecular Physiology
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Articles by Susan M. Majka in JoVE
JoVE General
अलगाव और Hoechst की विशेषता कम नकारात्मक फेफड़े mesenchymal स्टेम सेल माउस
1Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, 2Department of Medicine, University of Colorado Denver, 3Cancer Center, University of Colorado Denver, 4Webb Waring Institute, University of Colorado Denver
इस अनुच्छेद में हम murine निवासी फेफड़ों mesenchymal स्टेम कोशिकाओं (फेफड़ों एमएससी), उनके विस्तार, लक्षण और immunomodulatory गुणों का विश्लेषण के अलगाव को प्रदर्शित करता है.
Other articles by Susan M. Majka on PubMed
Stem Cells: a Minireview
The identification of adult-derived stem cells which maintain plasticity throughout the course of a lifetime, has transformed the field of stem cell biology. Bone marrow derived hematopoietic stem cells (HSC) are the most well-characterized population of these multipotential cells. First identified for their ability to reconstitute blood lineages and rescue lethally irradiated hosts, these cells have also been shown to differentiate and integrate into skeletal muscle, cardiac myocytes, vascular endothelium, liver, and brain tissue. Various populations of HSC are being studied, exploiting cell surface marker expression, such as Sca-1, c-kit, CD34, and lin; as well as the abilityto efflux the vital dye Hoecsht 33342. Detection of engrafted donor derived cells into various tissue types in vivo is a laborious process and may involve detection of beta-galactosidase via colorimetric reaction or antibody labeling or green fluorescent protein (GFP) via fluorescence microscopy, as well as in situ hybridization to detect the Y-chromosome. Using these techniques, the search has begun for tissue specific stem cells capable of host tissue regeneration, self renewal, and transdifferentiation. Caution is urged when interpreting these types of experiments because although they are stimulating, limitations of the technologies may provide misleading results.
Distinct Progenitor Populations in Skeletal Muscle Are Bone Marrow Derived and Exhibit Different Cell Fates During Vascular Regeneration
Vascular progenitors were previously isolated from blood and bone marrow; herein, we define the presence, phenotype, potential, and origin of vascular progenitors resident within adult skeletal muscle. Two distinct populations of cells were simultaneously isolated from hindlimb muscle: the side population (SP) of highly purified hematopoietic stem cells and non-SP cells, which do not reconstitute blood. Muscle SP cells were found to be derived from, and replenished by, bone marrow SP cells; however, within the muscle environment, they were phenotypically distinct from marrow SP cells. Non-SP cells were also derived from marrow stem cells and contained progenitors with a mesenchymal phenotype. Muscle SP and non-SP cells were isolated from Rosa26 mice and directly injected into injured muscle of genetically matched recipients. SP cells engrafted into endothelium during vascular regeneration, and non-SP cells engrafted into smooth muscle. Thus, distinct populations of vascular progenitors are resident within skeletal muscle, are derived from bone marrow, and exhibit different cell fates during injury-induced vascular regeneration.
Altered Phenotype and Reduced Function of Muscle-derived Hematopoietic Stem Cells
Skeletal muscle-derived cells have the potential to repopulate the major peripheral blood lineages of lethally irradiated mice and thus behave like hematopoietic stem cells (HSC). We have recently shown that muscle cells with HSC activity (ms-HSC) express CD45 and Sca-1, suggesting a hematopoietic origin. Here we sought to clarify contradictions in the literature regarding the phenotype of ms-HSC and precisely define the hematopoietic origin of these cells.
Identification of Novel Resident Pulmonary Stem Cells: Form and Function of the Lung Side Population
Resident lung stem cells function to replace all lineages of pulmonary tissue, including mesenchyme, epithelium, and vasculature. The phenotype of the lung side population (SP) cells is currently under investigation; their function is currently unknown. Recent data suggest lung SP cells are an enriched tissue-specific source of organ-specific pulmonary precursors and, therefore, a source of adult stem cells. The adult lung SP cell population has been isolated and characterized for expression of markers indicative of stem cell, epithelial, and mesenchymal lineages. These studies determined that the adult mouse lung SP has epithelial and mesenchymal potential that resides within a CD45- mesenchymal subpopulation, as well as limited hematopoietic ability, which resides in the bone marrow-derived CD45+ subpopulation. The ability to identify these adult lung precursor cells allows us to further study the potential of these cells and their role in the regulation of tissue homeostasis and response to injury. The identification of this target population will potentially allow earlier treatment and, long term, a functional restoration of injured pulmonary tissue and lung health.
Depletion of CAMP-response Element-binding Protein/ATF1 Inhibits Adipogenic Conversion of 3T3-L1 Cells Ectopically Expressing CCAAT/enhancer-binding Protein (C/EBP) Alpha, C/EBP Beta, or PPAR Gamma 2
The differentiation of preadipocytes to adipocytes is orchestrated by the expression of the "master adipogenic regulators," CCAAT/enhancer-binding protein (C/EBP) beta, peroxisome proliferator-activated receptor gamma (PPARgamma), and C/EBP alpha. In addition, activation of the cAMP-response element-binding protein (CREB) is necessary and sufficient to promote adipogenic conversion and prevent apoptosis of mature adipocytes. In this report we used small interfering RNA to deplete CREB and the closely related factor ATF1 to explore the ability of the master adipogenic regulators to promote adipogenesis in the absence of CREB and probe the function of CREB in late stages of adipogenesis. Loss of CREB/ATF1 blocked adipogenic conversion of 3T3-L1 cells in culture or 3T3-F442A cells implanted into athymic mice. Loss of CREB/ATF1 prevented the expression of PPARgamma, C/EBP alpha, and adiponectin and inhibited the loss of Pref-1. Loss of CREB/ATF1 inhibited adipogenic conversion even in cells ectopically expressing C/EBP alpha, C/EBP beta, or PPARgamma2 individually. CREB/ATF1 depletion did not attenuate lipid accumulation in cells expressing both PPARgamma2 and C/EBP alpha, but adiponectin expression was severely diminished. Conversely ectopic expression of constitutively active CREB overcame the blockade of adipogenesis due to depletion of C/EBP beta but not due to loss of PPARgamma2 or C/EBP alpha. Depletion of CREB/ATF1 did not suppress the expression of C/EBP beta as we had previously observed using dominant negative forms of CREB. Finally results are presented showing that CREB promotes PPARgamma2 gene transcription. The results indicate that CREB and ATF1 play a central role in adipogenesis because expression of individual master adipogenic regulators is unable to compensate for their loss. The data also indicate that CREB not only functions during the initiation of adipogenic conversion but also at later stages.
Rosiglitazone Promotes Development of a Novel Adipocyte Population from Bone Marrow-derived Circulating Progenitor Cells
Obesity and weight gain are characterized by increased adipose tissue mass due to an increase in the size of individual adipocytes and the generation of new adipocytes. New adipocytes are believed to arise from resident adipose tissue preadipocytes and mesenchymal progenitor cells. However, it is possible that progenitor cells from other tissues, in particular BM, could also contribute to development of new adipocytes in adipose tissue. We tested this hypothesis by transplanting whole BM cells from GFP-expressing transgenic mice into wild-type C57BL/6 mice and subjecting them to a high-fat diet or treatment with the thiazolidinedione (TZD) rosiglitazone (ROSI) for several weeks. Histological examination of adipose tissue or FACS of adipocytes revealed the presence of GFP(+) multilocular (ML) adipocytes, whose number was significantly increased by ROSI treatment or high-fat feeding. These ML adipocytes expressed adiponectin, perilipin, fatty acid-binding protein (FABP), leptin, C/EBPalpha, and PPARgamma but not uncoupling protein-1 (UCP-1), the CD45 hematopoietic lineage marker, or the CDllb monocyte marker. They also exhibited increased mitochondrial content. Appearance of GFP(+) ML adipocytes was contemporaneous with an increase in circulating levels of mesenchymal and hematopoietic progenitor cells in ROSI-treated animals. We conclude that TZDs and high-fat feeding promote the trafficking of BM-derived circulating progenitor cells to adipose tissue and their differentiation into ML adipocytes.
Syndecan-4-expressing Muscle Progenitor Cells in the SP Engraft As Satellite Cells During Muscle Regeneration
Skeletal muscle satellite cells, located between the basal lamina and plasma membrane of myofibers, are required for skeletal muscle regeneration. The capacity of satellite cells as well as other cell lineages including mesoangioblasts, mesenchymal stem cells, and side population (SP) cells to contribute to muscle regeneration has complicated the identification of a satellite stem cell. We have characterized a rare subset of the muscle SP that efficiently engrafts into the host satellite cell niche when transplanted into regenerating muscle, providing 75% of the satellite cell population and 30% of the myonuclear population, respectively. These cells are found in the satellite cell position, adhere to isolated myofibers, and spontaneously undergo myogenesis in culture. We propose that this subset of SP cells (satellite-SP cells), characterized by ABCG2, Syndecan-4, and Pax7 expression, constitutes a self-renewing muscle stem cell capable of generating both satellite cells and their myonuclear progeny in vivo.
De Novo Generation of White Adipocytes from the Myeloid Lineage Via Mesenchymal Intermediates is Age, Adipose Depot, and Gender Specific
It is generally assumed that white adipocytes arise from resident adipose tissue mesenchymal progenitor cells. We challenge this paradigm by defining a hematopoietic origin for both the de novo development of a subset of white adipocytes in adults and a previously uncharacterized adipose tissue resident mesenchymal progenitor population. Lineage and cytogenetic analysis revealed that bone marrow progenitor (BMP)-derived adipocytes and adipocyte progenitors arise from hematopoietic cells via the myeloid lineage in the absence of cell fusion. Global gene expression analysis indicated that the BMP-derived fat cells are bona fide adipocytes but differ from conventional white or brown adipocytes in decreased expression of genes involved in mitochondrial biogenesis and lipid oxidation, and increased inflammatory gene expression. The BMP-derived adipocytes accumulate with age, occur in higher numbers in visceral than in subcutaneous fat, and in female versus male mice. BMP-derived adipocytes may, therefore, account in part for adipose depot heterogeneity and detrimental changes in adipose metabolism and inflammation with aging and adiposity.
Osteoblasts Derived from Induced Pluripotent Stem Cells Form Calcified Structures in Scaffolds Both in Vitro and in Vitro
Reprogramming somatic cells into an embryonic stem (ES) cell-like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for eight to twelve weeks. Differentiation was assessed by lineage specific morphology, gene expression, histological stain and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic ICR mice. Implants were harvested at 12 weeks, and histological analyses of cell, mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage and fat was confirmed by morphology, and expression of lineage specific genes. Isolated implants of iPS cell derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells which merit evaluation for their potential uses in orthopaedic medicine and understanding of molecular mechanisms of orthopaedic disease.
The Pathology of Bleomycin-induced Fibrosis is Associated with Loss of Resident Lung Mesenchymal Stem Cells That Regulate Effector T-cell Proliferation
Tissue-resident mesenchymal stem cells (MSCs) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis, and tumor formation. Here, we define a population of resident lung MSCs (luMSCs) that function to regulate the severity of bleomycin injury via modulation of the T-cell response. Bleomycin-induced loss of these endogenous luMSCs and elicited fibrosis (pulmonary fibrosis), inflammation, and pulmonary arterial hypertension (PAH). Replacement of resident stem cells by administration of isolated luMSCs attenuated the bleomycin-associated pathology and mitigated the development of PAH. In addition, luMSC modulated a decrease in numbers of lymphocytes and granulocytes in bronchoalveolar fluid and demonstrated an inhibition of effector T-cell proliferation in vitro. Global gene expression analysis indicated that the luMSCs are a unique stromal population differing from lung fibroblasts in terms of proinflammatory mediators and profibrotic pathways. Our results demonstrate that luMSCs function to protect lung integrity after injury; however, when endogenous MSCs are lost, this function is compromised illustrating the importance of this novel population during lung injury. The definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury.
Concise Review: Adipocyte Origins: Weighing the Possibilities
Adipose tissue is the primary energy reservoir in the body and an important endocrine organ that plays roles in energy homeostasis, feeding, insulin sensitivity, and inflammation. While it was tacitly assumed that fat in different anatomical locations had a common origin and homogenous function, it is now clear that regional differences exist in adipose tissue characteristics and function. This is exemplified by the link between increased deep abdominal or visceral fat, but not peripheral adipose tissue and the metabolic disturbances associated with obesity. Regional differences in fat function are due in large part to distinct adipocyte populations that comprise the different fat depots. Evidence accrued primarily in the last decade indicates that the distinct adipocyte populations are generated by a number of processes during and after development. These include the production of adipocytes from different germ cell layers, the formation of distinct preadipocyte populations from mesenchymal progenitors of mesodermal origin, and the production of adipocytes from hematopoietic stem cells from the bone marrow. This review will examine each of these process and their relevance to normal adipose tissue formation and contribution to obesity-related diseases.
Reduction of Reactive Oxygen Species Prevents Hypoxia-induced CREB Depletion in Pulmonary Artery Smooth Muscle Cells
Hypoxia-induced pulmonary arterial hypertension (PAH) is a deadly disease characterized by progressive remodeling and persistent vasoconstriction of the pulmonary arterial system. Remodeling of the pulmonary artery (PA) involves smooth muscle cell (SMC) proliferation, hypertrophy, migration, and elevated extracellular matrix (ECM) production elicited by mitogens and oxidants produced in response to hypoxic insult. We previously reported that the transcription factor cAMP response element binding protein (CREB) is depleted in medial PA SMCs in remodeled, hypertensive vessels in rats or calves exposed to chronic hypoxia. In culture, CREB loss can be induced in PA SMCs by exogenous oxidants or platelet-derived growth factor. Forced depletion of CREB with small interfering RNA (siRNA) in PA SMCs is sufficient to induce their proliferation, hypertrophy, migration, dedifferentiation, and ECM production. This suggests that oxidant and/or mitogen-induced loss of CREB in medial SMCs is, in part, responsible for PA thickening. Here, we tested whether oxidant scavengers could prevent the loss of CREB in PA SMCs and inhibit SMC proliferation, migration, and ECM production using in vitro and in vivo models. Exposure of PA SMCs to hypoxia induced hydrogen peroxide (H2O2) production and loss of CREB. Treatment of SMCs with exogenous H2O2 or a second oxidant, Sin-1, elicited CREB depletion under normoxic conditions. Exogenous H2O2 also induced SMC proliferation, migration, and increased elastin levels as did forced depletion of CREB. In vivo, hypoxia-induced thickening of the PA wall was suppressed by the superoxide dismutase mimetic, Tempol, which also prevented the loss of CREB in medial SMCs. Tempol also reduced hypoxia-induced SMC proliferation and elastin deposition in the PA. The data indicate that CREB levels in the arterial wall are regulated in part by oxidants produced in response to hypoxia and that CREB plays a crucial role in regulating SMC phenotype and PA remodeling.
Osteoblasts Derived from Induced Pluripotent Stem Cells Form Calcified Structures in Scaffolds Both in Vitro and in Vivo
Reprogramming somatic cells into an ESC-like state, or induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies. In this study, we performed directed differentiation to assess the ability of murine iPS cells to differentiate into bone, cartilage, and fat in vitro and to maintain an osteoblast phenotype on a scaffold in vitro and in vivo. Embryoid bodies derived from murine iPS cells were cultured in differentiation medium for 8–12 weeks. Differentiation was assessed by lineage-specific morphology, gene expression, histological stain, and immunostaining to detect matrix deposition. After 12 weeks of expansion, iPS-derived osteoblasts were seeded in a gelfoam matrix followed by subcutaneous implantation in syngenic imprinting control region (ICR) mice. Implants were harvested at 12 weeks, histological analyses of cell and mineral and matrix content were performed. Differentiation of iPS cells into mesenchymal lineages of bone, cartilage, and fat was confirmed by morphology and expression of lineage-specific genes. Isolated implants of iPS cell-derived osteoblasts expressed matrices characteristic of bone, including osteocalcin and bone sialoprotein. Implants were also stained with alizarin red and von Kossa, demonstrating mineralization and persistence of an osteoblast phenotype. Recruitment of vasculature and microvascularization of the implant was also detected. Taken together, these data demonstrate functional osteoblast differentiation from iPS cells both in vitro and in vivo and reveal a source of cells, which merit evaluation for their potential uses in orthopedic medicine and understanding of molecular mechanisms of orthopedic disease.
Programmatic Change: Lung Disease Research in the Era of Induced Pluripotency
Human lung research has made remarkable progress over the last century largely through the use of animal models of disease. The challenge for the future is to translate these findings into human disease and bring about meaningful disease modification or even cure. The ability to generate transformative therapies in the future will require human tissue, currently scarce under the best of circumstances. Unfortunately, patient-derived somatic cells are often poorly characterized and have a limited life span in culture. Moreover, these cells are frequently obtained from patients with end-stage disease exposed to multiple drug therapies, leaving researchers with questions about whether their findings recapitulate disease-initiating processes or are simply the result of pharmacological intervention or subsequent host responses. The goal of studying early disease in multiple cell and tissue types has driven interest in the use of induced pluripotent stem cells (iPSCs) to model lung disease. These cells provide an alternative model for relevant lung research and hold promise in particular for studying the initiation of disease processes in genetic conditions such as heritable pulmonary arterial hypertension as well as other lung diseases. In this Perspective, we focus on potential iPSC use in pulmonary vascular disease research as a model for iPSC use in many types of advanced lung disease.
