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1.
Ann Thorac Surg ; 2019 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-31526779

RESUMO

BACKGROUND: Bone marrow stromal/stem cells (BMSCs) remain a promising potential therapy for ischemic cardiomyopathy. The primary objective of this study was to evaluate the safety and feasibility of direct intramyocardial injection of autologous BMSCs in patients undergoing transmyocardial revascularization (TMR) or coronary artery bypass graft surgery (CABG). METHODS: A phase I trial was conducted on adult patients suffering from ischemic heart disease with depressed left ventricular ejection fraction who were scheduled to undergo TMR or CABG. Autologous BMSCs were expanded for three weeks prior to scheduled surgery. Following completion of surgical revascularization, BMSCs were directly injected into ischemic myocardium. Safety and feasibility of therapy were assessed. Cardiac functional status and changes in quality of life were evaluated at one year. RESULTS: Fourteen patients underwent simultaneous BMSC and surgical revascularization therapy (TMR+BMSCs = 10, CABG+BMSCs = 4). BMSCs were successfully expanded and no significant complications occurred as a result of the procedure. Regional contractility in the cell-treated areas demonstrated improvement at 12 months compared to baseline (TMR+BMSCs Δ strain: -4.6% ± 2.1%; P = 0.02. CABG+MSCs Δ strain: -4.2% ± 6.0%; P = 0.30). Quality of life was enhanced, with substantial reduction in angina scores at one year post-treatment (TMR+BMSCs: 1.3 ± 1.2. CABG+MSCs: 1.0 ± 1.4). CONCLUSIONS: In this phase 1 trial, direct intramyocardial injection of autologous BMSCs in conjunction with TMR or CABG was technically feasible and could be performed safely. Preliminary results demonstrate improved cardiac function and quality of life in patients at one year after treatment.

2.
J Bone Miner Res ; 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31545883

RESUMO

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2019 American Society for Bone and Mineral Research.

3.
Am J Hum Genet ; 104(5): 925-935, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982609

RESUMO

Colony stimulating factor 1 receptor (CSF1R) plays key roles in regulating development and function of the monocyte/macrophage lineage, including microglia and osteoclasts. Mono-allelic mutations of CSF1R are known to cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an adult-onset progressive neurodegenerative disorder. Here, we report seven affected individuals from three unrelated families who had bi-allelic CSF1R mutations. In addition to early-onset HDLS-like neurological disorders, they had brain malformations and skeletal dysplasia compatible to dysosteosclerosis (DOS) or Pyle disease. We identified five CSF1R mutations that were homozygous or compound heterozygous in these affected individuals. Two of them were deep intronic mutations resulting in abnormal inclusion of intron sequences in the mRNA. Compared with Csf1r-null mice, the skeletal and neural phenotypes of the affected individuals appeared milder and variable, suggesting that at least one of the mutations in each affected individual is hypomorphic. Our results characterized a unique human skeletal phenotype caused by CSF1R deficiency and implied that bi-allelic CSF1R mutations cause a spectrum of neurological and skeletal disorders, probably depending on the residual CSF1R function.

4.
Stem Cells Transl Med ; 8(6): 586-592, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30767420

RESUMO

Osteoarthritic and other types of articular cartilage defects never heal on their own. Medicinal and surgical approaches are often ineffective, and the supply of autologous chondrocytes for tissue engineering is very limited. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested as an adequate cell source for cartilage reconstruction. However, the majority of studies employing BMSCs for cartilage tissue engineering have used BMSCs predifferentiated into cartilage prior to implantation. This strategy has failed to achieve formation of stable, hyaline-like cartilage, resistant to hypertrophy in vivo. We hypothesized that in vitro predifferentiation of BMSCs is not necessary when cells are combined with an adequate scaffold that supports the formation of stable cartilage in vivo. In this study, naïve (undifferentiated) human BMSCs were attached to dehydrothermally crosslinked stable fibrin microbeads (FMBs) without and with other scaffolds and implanted subcutaneously into immunocompromised mice. Optimal formation of abundant, hypertrophy-resistant, ectopic hyaline-like cartilage was achieved when BMSCs were attached to FMBs covalently coated with hyaluronic acid. The cartilage that was formed was of human origin and was stable for at least 28 weeks in vivo. Stem Cells Translational Medicine 2019;8:586-592.

5.
J Bone Miner Res ; 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30496606

RESUMO

Fibrous dysplasia of bone (FD) is a mosaic disease caused by mutations in GNAS. Constitutive activation of the α-subunit of the Gs stimulatory protein (Gαs) leads to dysregulated proliferation of bone marrow stromal cells (BMSCs), generating expansile lesions of fibrotic tissue and abnormal bone. Local bone remodeling regulation by BMSCs is also altered, and FD tissue is characterized by abundant osteoclast-like cells that may be essential for lesion expansion. Animal models show local expression of RANKL in bone lesions, and treatment with the RANKL neutralizing antibody denosumab decreased lesion expansion rate in a patient with aggressive FD. However, the role of RANKL/osteoprotegerin (OPG) in FD pathophysiology is not yet understood. We measured serum levels of RANKL, OPG, and inactive RANKL-OPG complexes in FD patients of known disease burden and in healthy volunteers (HVs). RANK, RANKL, and Ki67 immunohistochemistry were assessed in FD tissue. Cultured FD and HV BMSCs were stimulated with prostaglandin E2 (PGE2 ) and 1,25 vitamin D3 to increase RANKL expression, and media levels of RANKL and OPG were measured. Osteoclastogenic induction by FD or HV BMSCs was assessed in co-cultures with HV peripheral monocytes. FD patients showed a 16-fold increase in serum RANKL compared to HVs. OPG was moderately increased (24%), although RANKL/OPG ratio was 12-fold higher in FD patients than in HVs. These measurements were positively correlated with the skeletal burden score (SBS), a validated marker of overall FD burden. No differences in serum inactive RANKL-OPG complexes were observed. In FD tissue, RANKL+ and Ki67+ fibroblastic cells were observed near RANK+ osteoclasts. High levels of RANKL were released by FD BMSCs cultures, but were undetectable in HV cultures. FD BMSC released less OPG than HV BMSCs. FD, but not HV BMSCs, induced osteoclastogenesis in monocyte co-cultures, which was prevented by denosumab addition. These data are consistent with the role of RANKL as a driver in FD-induced osteoclastogenesis. © 2018 American Society for Bone and Mineral Research.

6.
Cytotherapy ; 20(11): 1381-1400, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30316562

RESUMO

The Signature Series Symposium "Cellular Therapies for Orthopaedics and Musculoskeletal Disease Proven and Unproven Therapies-Promise, Facts and Fantasy" was held as a pre-meeting of the 26th International Society for Cellular Therapy (ISCT) annual congress in Montreal, Canada, May 2, 2018. This was the first ISCT program that was entirely dedicated to the advancement of cell-based therapies for musculoskeletal diseases. Cellular therapies in musculoskeletal medicine are a source of great promise and opportunity. They are also the source of public controversy, confusion and misinformation. Patients, clinicians, scientists, industry and government share a commitment to clear communication and responsible development of the field. Therefore, this symposium convened thought leaders from around the world in a forum designed to catalyze communication and collaboration to bring the greatest possible innovation and value to patients with musculoskeletal conditions.

7.
Biomaterials ; 186: 31-43, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30278344

RESUMO

Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (µCT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). Combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs.

8.
Nature ; 561(7724): 455-457, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30258150
9.
J Bone Miner Res ; 33(10): 1721-1728, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30133922

RESUMO

Cell-based therapies hold much promise for musculoskeletal medicine; however, this rapidly growing field faces a number of challenges. Few of these therapies have proven clinical benefit, and an insufficient regulatory environment has allowed for widespread clinical implementation without sufficient evidence of efficacy. The technical and biological complexity of cell-based therapies has contributed to difficulties with reproducibility and mechanistic clarity. In order to aid in addressing these challenges, we aim to clarify the key issues in the preclinical cell therapy field, and to provide a conceptual framework for advancing the state of the science. Broadly, these suggestions relate to: (i) delineating cell-therapy types and moving away from "catch-all" terms such as "stem cell" therapies; (ii) clarifying descriptions of cells and their processing; and (iii) increasing the standard of in vivo evaluation of cell-based therapy experiments to determining cell fates. Further, we provide an overview of methods for experimental evaluation, data sharing, and professional society participation that would be instrumental in advancing this field. © 2018 American Society for Bone and Mineral Research.

10.
Trends Mol Med ; 24(9): 805-820, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30006147

RESUMO

Use of human pluripotent stem cells (hPSCs) and their differentiated derivatives have led to recent proof-of-principle drug discoveries, defining a pathway to the implementation of hPSC-based drug discovery (hPDD). Current hPDD strategies, however, have inevitable conceptual biases and technological limitations, including the dimensionality of cell-culture methods, cell maturity and functionality, experimental variability, and data reproducibility. In this review, we dissect representative hPDD systems via analysis of hPSC-based 2D-monolayers, 3D culture, and organoids. We discuss mechanisms of drug discovery and drug repurposing, and roles of membrane drug transporters in tissue maturation and hPDD using the example of drugs that target various mutations of CFTR, the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis.

11.
J Transl Med ; 16(1): 65, 2018 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-29540180

RESUMO

BACKGROUND: Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture. METHODS: A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared. RESULTS: Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF. CONCLUSIONS: Traditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.

12.
Stem Cells ; 36(1): 11-21, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28948674

RESUMO

Lineage commitment and differentiation of skeletal stem cells/bone marrow stromal cells (SSCs/BMSCs, often called bone marrow-derived "mesenchymal stem/stromal" cells) offer an important opportunity to study skeletal and hematopoietic diseases, and for tissue engineering and regenerative medicine. Currently, many studies in this field have relied on cell lineage tracing methods in mouse models, which have provided a significant advancement in our knowledge of skeletal and hematopoietic stem-cell niches in bone marrow (BM). However, there is a lack of agreement in numerous fundamental areas, including origins of various BM stem-cell niches, cell identities, and their physiological roles in the BM. In order to resolve these issues, we propose a new hypothesis of "paralogous" stem-cell niches (PSNs); that is, progressively altered parallel niches within an individual species throughout the life span of the organism. A putative PSN code seems to be plausible based on analysis of transcriptional signatures in two representative genes that encode Nes-GFP and leptin receptors, which are frequently used to monitor SSC lineage development in BM. Furthermore, we suggest a dynamic paralogous BM niche (PBMN) model that elucidates the coupling and uncoupling mechanisms between BM stem-cell niches and their zones of active regeneration during different developmental stages. Elucidation of these PBMNs would enable us to resolve the existing controversies, thus paving the way to achieving precision regenerative medicine and pharmaceutical applications based on these BM cell resources. Stem Cells 2018;36:11-21.


Assuntos
Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Nicho de Células-Tronco/genética , Células-Tronco/metabolismo , Diferenciação Celular , Linhagem da Célula , Humanos
13.
Stem Cell Reports ; 9(5): 1343-1358, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29056332

RESUMO

The development of mouse genetic tools has made a significant contribution to the understanding of skeletal and hematopoietic stem cell niches in bone marrow (BM). However, many experimental designs (e.g., selections of marker genes, target vector constructions, and choices of reporter murine strains) have unavoidable technological limitations and bias, which lead to experimental discrepancies, data reproducibility issues, and frequent data misinterpretation. Consequently, there are a number of conflicting views relating to fundamental biological questions, including origins and locations of skeletal and hematopoietic stem cells in the BM. In this report, we systematically unravel complicated data interpretations via comprehensive analyses of technological benefits, pitfalls, and challenges in frequently used mouse models and discuss their translational relevance to human stem cell biology. Particularly, we emphasize the important roles of using large human genomic data-informatics in facilitating genetic analyses of mouse models and resolving existing controversies in mouse and human BM stem cell biology.


Assuntos
Células da Medula Óssea/citologia , Genômica/métodos , Camundongos/genética , Modelos Animais , Nicho de Células-Tronco/genética , Pesquisa Médica Translacional/métodos , Animais , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genômica/normas , Pesquisa Médica Translacional/normas
14.
Skeletal Radiol ; 46(10): 1435-1439, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28660402

RESUMO

Bisphosphonates (BPs) are currently used in the treatment of diverse bone diseases including fibrous dysplasia of bone (FD). In pediatric patients, a radiographic consequence of cyclical administration of BPs is the development of apo-, epi-, and meta-physeal sclerotic bands, otherwise known as zebra lines, which result from the temporary inhibition of osteoclastic activity at the time of drug treatment. We report here on a child with McCune-Albright syndrome (FD in addition to hyperfunctioning endocrinopathies and skin hyperpigmentation) treated with cyclical intravenous infusions of pamidronate in which conventional radiography, contact microradiography, histology, and backscattered electron image analysis demonstrated that zebra lines formed only where bone was normal, were arrested at the boundary between FD-unaffected and FD-affected bone where bone is sclerotic, and were absent within the undermineralized FD bone. Moreover, in spite of the treatment, the FD lesions continued to expand. This case report is unique because no previously published studies correlated the radiographic and the histologic features of BP-induced zebra lines in the metaphysis of an FD-affected long bone of the limbs.


Assuntos
Difosfonatos/efeitos adversos , Fêmur/efeitos dos fármacos , Fêmur/diagnóstico por imagem , Displasia Fibrosa Poliostótica/diagnóstico por imagem , Displasia Fibrosa Poliostótica/tratamento farmacológico , Adolescente , Progressão da Doença , Feminino , Displasia Fibrosa Poliostótica/cirurgia , Humanos , Pamidronato
15.
Sci Rep ; 7: 46731, 2017 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-28447618

RESUMO

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

16.
Development ; 144(6): 1035-1044, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28292847

RESUMO

Human umbilical cord blood (CB) has attracted much attention as a reservoir for functional hematopoietic stem and progenitor cells, and, recently, as a source of blood-borne fibroblasts (CB-BFs). Previously, we demonstrated that bone marrow stromal cell (BMSC) and CB-BF pellet cultures make cartilage in vitro Furthermore, upon in vivo transplantation, BMSC pellets remodelled into miniature bone/marrow organoids. Using this in vivo model, we asked whether CB-BF populations that express characteristics of the hematopoietic stem cell (HSC) niche contain precursors that reform the niche. CB ossicles were regularly observed upon transplantation. Compared with BM ossicles, CB ossicles showed a predominance of red marrow over yellow marrow, as demonstrated by histomorphological analyses and the number of hematopoietic cells isolated within ossicles. Marrow cavities from CB and BM ossicles included donor-derived CD146-expressing osteoprogenitors and host-derived mature hematopoietic cells, clonogenic lineage-committed progenitors and HSCs. Furthermore, human CD34+ cells transplanted into ossicle-bearing mice engrafted and maintained human HSCs in the niche. Our data indicate that CB-BFs are able to recapitulate the conditions by which the bone marrow microenvironment is formed and establish complete HSC niches, which are functionally supportive of hematopoietic tissue.


Assuntos
Células da Medula Óssea/citologia , Sangue Fetal/citologia , Fibroblastos/citologia , Células-Tronco Hematopoéticas/citologia , Organoides/citologia , Nicho de Células-Tronco , Adulto , Compartimento Celular , Criança , Fibroblastos/transplante , Transplante de Células-Tronco Hematopoéticas , Homeostase , Humanos , Nicho de Células-Tronco/genética , Células Estromais/citologia
17.
Stem Cell Reports ; 6(6): 897-913, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27304917

RESUMO

A widely shared view reads that mesenchymal stem/stromal cells ("MSCs") are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as "MSCs" based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called "MSCs," with important applicative implications. The data also support the view that rather than a uniform class of "MSCs," different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin.


Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Microvasos/citologia , Pericitos/citologia , Células Satélites de Músculo Esquelético/citologia , Transcriptoma , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/metabolismo , Diferenciação Celular , Linhagem da Célula/genética , Condrogênese/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Microvasos/metabolismo , Osteogênese/genética , Pericitos/metabolismo , Fenótipo , Células Satélites de Músculo Esquelético/metabolismo , Transplante Heterólogo
18.
PLoS One ; 10(11): e0142554, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26565809

RESUMO

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 µg/µg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 µg/µg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.


Assuntos
Glicogênio/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo
19.
Bonekey Rep ; 4: 654, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26229595

RESUMO

The cellular mechanisms involved in the asymmetric facial overgrowth syndrome, hemifacial hyperplasia (HFH), are not well understood. This study was conducted to compare primary cell cultures from hyperplastic and normal HFH bone for cellular and molecular differences. Primary cultures developed from biopsies of a patient with isolated HFH showed a twofold difference in cell size and cell number between hyperplastic and normal bone. Microarray data suggested a 40% suppression of PTEN (phosphatase-tensin homolog) transcripts. Sequencing of the PTEN gene and promoter identified novel C/G missense mutation (position -1053) in the regulatory region of the PTEN promoter. Western blots of downstream pathway components showed an increase in PKBa/Akt1 phosphorylation and TOR (target of rapamcyin) signal. Sirolimus, an inhibitor of TOR, when added to overgrowth cells reversed the cell size, cell number and total protein differences between hyperplastic and normal cells. In cases of facial overgrowth, which involve PTEN/Akt/TOR dysregulation, sirolimus could be used for limiting cell overgrowth.

20.
Stem Cell Res ; 15(1): 42-53, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26001169

RESUMO

Patients with systemic mastocytosis (SM) have a wide variety of problems, including skeletal abnormalities. The disease results from a mutation of the stem cell receptor (c-kit) in mast cells and we wondered if the function of bone marrow stromal cells (BMSCs; also known as MSCs or mesenchymal stem cells) might be affected by the invasion of bone marrow by mutant mast cells. As expected, BMSCs from SM patients do not have a mutation in c-kit, but they proliferate poorly. In addition, while osteogenic differentiation of the BMSCs seems to be deficient, their adipogenic potential appears to be increased. Since the hematopoietic supportive abilities of BMSCs are also important, we also studied the engraftment in NSG mice of human CD34(+) hematopoietic progenitors, after being co-cultured with BMSCs of healthy volunteers vs. BMSCs derived from patients with SM. BMSCs derived from the bone marrow of patients with SM could not support hematopoiesis to the extent that healthy BMSCs do. Finally, we performed an expression analysis and found significant differences between healthy and SM derived BMSCs in the expression of genes with a variety of functions, including the WNT signaling, ossification, and bone remodeling. We suggest that some of the symptoms associated with SM might be driven by epigenetic changes in BMSCs caused by dysfunctional mast cells in the bone marrow of the patients.


Assuntos
Células da Medula Óssea/patologia , Mastocitose Sistêmica/patologia , Adipogenia/genética , Adulto , Idoso , Animais , Estudos de Casos e Controles , Proliferação de Células , Forma Celular , Ensaio de Unidades Formadoras de Colônias , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Hematopoese/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação/genética , Osteogênese/genética , Proteínas Proto-Oncogênicas c-kit/genética , Células Estromais/patologia , Doadores de Tecidos
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