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1.
Am J Physiol Cell Physiol ; 324(4): C821-C836, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36802732

RESUMO

Pericytes are mural cells that play an important role in regulation of angiogenesis and endothelial function. Cadherins are a superfamily of adhesion molecules mediating Ca2+-dependent homophilic cell-cell interactions that control morphogenesis and tissue remodeling. To date, classical N-cadherin is the only cadherin described on pericytes. Here, we demonstrate that pericytes also express T-cadherin (H-cadherin, CDH13), an atypical glycosyl-phosphatidylinositol (GPI)-anchored member of the superfamily that has previously been implicated in regulation of neurite guidance, endothelial angiogenic behavior, and smooth muscle cell differentiation and progression of cardiovascular disease. The aim of the study was to investigate T-cadherin function in pericytes. Expression of T-cadherin in pericytes from different tissues was performed by immunofluorescence analysis. Using lentivirus-mediated gain-of-function and loss-of-function in cultured human pericytes, we demonstrate that T-cadherin regulates pericyte proliferation, migration, invasion, and interactions with endothelial cells during angiogenesis in vitro and in vivo. T-cadherin effects are associated with the reorganization of the cytoskeleton, modulation of cyclin D1, α-smooth muscle actin (αSMA), integrin ß3, metalloprotease MMP1, and collagen expression levels, and involve Akt/GSK3ß and ROCK intracellular signaling pathways. We also report the development of a novel multiwell 3-D microchannel slide for easy analysis of sprouting angiogenesis from a bioengineered microvessel in vitro. In conclusion, our data identify T-cadherin as a novel regulator of pericyte function and support that it is required for pericyte proliferation and invasion during active phase of angiogenesis, while T-cadherin loss shifts pericytes toward the myofibroblast state rendering them unable to control endothelial angiogenic behavior.


Assuntos
Células Endoteliais , Pericitos , Humanos , Pericitos/metabolismo , Células Endoteliais/metabolismo , Caderinas/genética , Caderinas/metabolismo , Morfogênese , Neovascularização Fisiológica
2.
Proc Natl Acad Sci U S A ; 115(18): 4625-4630, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29666250

RESUMO

It is generally accepted that adult human bone marrow-derived mesenchymal stromal cells (hMSCs) are default committed toward osteogenesis. Even when induced to chondrogenesis, hMSCs typically form hypertrophic cartilage that undergoes endochondral ossification. Because embryonic mesenchyme is obviously competent to generate phenotypically stable cartilage, it is questioned whether there is a correspondence between mesenchymal progenitor compartments during development and in adulthood. Here we tested whether forcing specific early events of articular cartilage development can program hMSC fate toward stable chondrogenesis. Inspired by recent findings that spatial restriction of bone morphogenetic protein (BMP) signaling guides embryonic progenitors toward articular cartilage formation, we hypothesized that selective inhibition of BMP drives the phenotypic stability of hMSC-derived chondrocytes. Two BMP type I receptor-biased kinase inhibitors were screened in a microfluidic platform for their time- and dose-dependent effect on hMSC chondrogenesis. The different receptor selectivity profile of tested compounds allowed demonstration that transient blockade of both ALK2 and ALK3 receptors, while permissive to hMSC cartilage formation, is necessary and sufficient to maintain a stable chondrocyte phenotype. Remarkably, even upon compound removal, hMSCs were no longer competent to undergo hypertrophy in vitro and endochondral ossification in vivo, indicating the onset of a constitutive change. Our findings demonstrate that adult hMSCs effectively share properties of embryonic mesenchyme in the formation of transient but also of stable cartilage. This opens potential pharmacological strategies to articular cartilage regeneration and more broadly indicates the relevance of developmentally inspired protocols to control the fate of adult progenitor cell systems.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Engenharia Tecidual/métodos , Receptores de Ativinas Tipo I/metabolismo , Adulto , Animais , Células da Medula Óssea/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Condrogênese/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteogênese/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos
3.
Int J Mol Sci ; 21(19)2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-33008121

RESUMO

Most bones of the human body form and heal through endochondral ossification, whereby hypertrophic cartilage (HyC) is formed and subsequently remodeled into bone. We previously demonstrated that HyC can be engineered from human mesenchymal stromal cells (hMSC), and subsequently devitalized by apoptosis induction. The resulting extracellular matrix (ECM) tissue retained osteoinductive properties, leading to ectopic bone formation. In this study, we aimed at engineering and devitalizing upscaled quantities of HyC ECM within a perfusion bioreactor, followed by in vivo assessment in an orthotopic bone repair model. We hypothesized that the devitalized HyC ECM would outperform a clinical product currently used for bone reconstructive surgery. Human MSC were genetically engineered with a gene cassette enabling apoptosis induction upon addition of an adjuvant. Engineered hMSC were seeded, differentiated, and devitalized within a perfusion bioreactor. The resulting HyC ECM was subsequently implanted in a 10-mm rabbit calvarial defect model, with processed human bone (Maxgraft®) as control. Human MSC cultured in the perfusion bioreactor generated a homogenous HyC ECM and were efficiently induced towards apoptosis. Following six weeks of in vivo implantation, microcomputed tomography and histological analyses of the defects revealed an increased bone formation in the defects filled with HyC ECM as compared to Maxgraft®. This work demonstrates the suitability of engineered devitalized HyC ECM as a bone substitute material, with a performance superior to a state-of-the-art commercial graft. Streamlined generation of the devitalized tissue transplant within a perfusion bioreactor is relevant towards standardized and automated manufacturing of a clinical product.


Assuntos
Cartilagem/crescimento & desenvolvimento , Diferenciação Celular/genética , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Crânio/crescimento & desenvolvimento , Animais , Apoptose/genética , Remodelação Óssea/genética , Substitutos Ósseos/uso terapêutico , Cartilagem/metabolismo , Cartilagem/transplante , Matriz Extracelular/genética , Humanos , Transplante de Células-Tronco Mesenquimais , Osteogênese/genética , Coelhos , Crânio/fisiopatologia , Crânio/cirurgia , Engenharia Tecidual/métodos , Alicerces Teciduais , Cicatrização/genética
4.
Proc Natl Acad Sci U S A ; 111(49): 17426-31, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25422415

RESUMO

The role of cell-free extracellular matrix (ECM) in triggering tissue and organ regeneration has gained increased recognition, yet current approaches are predominantly based on the use of ECM from fully developed native tissues at nonhomologous sites. We describe a strategy to generate customized ECM, designed to activate endogenous regenerative programs by recapitulating tissue-specific developmental processes. The paradigm was exemplified in the context of the skeletal system by testing the osteoinductive capacity of engineered and devitalized hypertrophic cartilage, which is the primordial template for the development of most bones. ECM was engineered by inducing chondrogenesis of human mesenchymal stromal cells and devitalized by the implementation of a death-inducible genetic device, leading to cell apoptosis on activation and matrix protein preservation. The resulting hypertrophic cartilage ECM, tested in a stringent ectopic implantation model, efficiently remodeled to form de novo bone tissue of host origin, including mature vasculature and a hematopoietic compartment. Importantly, cartilage ECM could not generate frank bone tissue if devitalized by standard "freeze & thaw" (F&T) cycles, associated with a significant loss of glycosaminoglycans, mineral content, and ECM-bound cytokines critically involved in inflammatory, vascularization, and remodeling processes. These results support the utility of engineered ECM-based devices as off-the-shelf regenerative niches capable of recruiting and instructing resident cells toward the formation of a specific tissue.


Assuntos
Apoptose , Cartilagem/fisiologia , Matriz Extracelular/fisiologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Adulto , Animais , Regeneração Óssea , Sistema Livre de Células , Feminino , Citometria de Fluxo , Congelamento , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Osteogênese , Microtomografia por Raio-X , Adulto Jovem
5.
Bioact Mater ; 24: 174-184, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36606254

RESUMO

The increasing recognition of the contribution of the immune system to activate and prime regeneration implies that tissue engineering strategies and biomaterials design should target regulation of early immunological processes. We previously proposed the cell-based engineering and devitalization of extracellular matrices (ECMs) as a strategy to generate implant materials delivering custom-defined signals. Here, in the context of bone regeneration, we aimed at enhancing the osteoinductivity of such ECMs by enriching their immunomodulatory factors repertoire. Priming with IL1ß a cell line overexpressing BMP-2 enabled engineering of ECMs preserving osteoinductive signals and containing larger amounts of angiogenic (VEGF) and pro-inflammatory molecules (IL6, IL8 and MCP1). Upon implantation, these IL1ß-induced materials enhanced processes typical of the inflammatory phase (e.g., vascular invasion, osteoclast recruitment and differentiation), leading to 'regenerative' events (e.g., M2 macrophage polarization) and ultimately resulting in faster and more efficient bone formation. These results bear relevance towards the manufacturing of potent off-the-shelf osteoinductive materials and outline the broader paradigm of engineering immunoinstructive implants to enhance tissue regeneration.

6.
Adv Healthc Mater ; 12(9): e2202550, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36527264

RESUMO

Engineering living bone tissue of defined shape on-demand has remained a challenge. 3D bioprinting (3DBP), a biofabrication process capable of yielding cell constructs of defined shape, when combined with developmental engineering can provide a possible path forward. Through the development of a bioink possessing appropriate rheological properties to carry a high cell load and concurrently yield physically stable structures, printing of stable, cell-laden, single-matrix constructs of anatomical shapes is realized without the need for fugitive or support phases. Using this bioink system, constructs of hypertrophic cartilage of predesigned geometry are engineered in vitro by printing human mesenchymal stromal cells at a high density to drive spontaneous condensation and implanted in nude mice to evoke endochondral ossification. The implanted constructs retain their prescribed shape over a 12-week period and undergo remodeling to yield ossicles of the designed shape with neovascularization. Microcomputed tomography, histological, and immunohistochemistry assessments confirm bone tissue characteristics and the presence of human cells. These results demonstrate the potential of 3DBP to fabricate complex bone tissue for clinical application.


Assuntos
Bioimpressão , Camundongos , Animais , Humanos , Bioimpressão/métodos , Camundongos Nus , Microtomografia por Raio-X , Engenharia Tecidual/métodos , Osso e Ossos , Alicerces Teciduais/química , Impressão Tridimensional
7.
Acta Biomater ; 154: 641-649, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36261107

RESUMO

We previously described an immortalized, genetically-engineered human Mesenchymal stromal cell line to generate BMP2-enriched Chondrogenic Matrices (MB-CM), which after devitalization and storage could efficiently induce ectopic bone tissue by endochondral ossification. In order to increase the efficiency of MB-CM utilization towards engineering scaled-up bone structures, here we hypothesized that MB-CM can retain osteoinductive properties when combined with an osteoconductive material. We first tested different volumetric ratios of MB-CM:SmartBone® (as clinically used, osteoconductive reference material) and assessed the bone formation capacity of the resulting composites following ectopic mouse implantation. After 8 weeks, as little as 25% of MB-CM was sufficient to induce bone formation and fusion across SmartBone® granules, generating large interconnected bony structures. The same composite percentage was then further assessed in a scaled-up model, namely within an axially-vascularized, confined, ectopically prefabricated flap (0.8 cm3) in rats. The material efficiently induced the formation of new bone (31% of the cross-sectional area after 8 weeks), including bone marrow and vascular elements, throughout the flap volume. Our findings outline a strategy for efficient use of MB-CM as part of a composite material, thereby reducing the amount required to fill large spaces and enabling utilization in critically sized grafts, to address challenging clinical scenarios in bone reconstruction. STATEMENT OF SIGNIFICANCE: Most bone repair strategies rely either on osteconductive properties of ceramics and devitalized bone, or osteoinductive properties of growth factors and extracellular matrices (ECM). Here we designed a composite material made of a clinically accepted osteoconductive material and an off-the-shelf tissue engineered human cartilage ECM with strong osteoinductive properties. We showed that low amount of osteoinductive ECM potentiated host cells recruitment to form large vascularized bone structures in two different animal models, one being a challenging prefabricated bone-flap model targeting challenging clinical bone repair. Overall, this study highlights the use of a promising human off-the-shelf material for accelerated healing towards clinical applications.


Assuntos
Osteogênese , Engenharia Tecidual , Ratos , Camundongos , Humanos , Animais , Engenharia Tecidual/métodos , Cartilagem , Regeneração Óssea , Condrogênese
8.
Stem Cells Transl Med ; 11(2): 213-229, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35259280

RESUMO

Cells of the stromal vascular fraction (SVF) of human adipose tissue have the capacity to generate osteogenic grafts with intrinsic vasculogenic properties. However, cultured adipose-derived stromal cells (ASCs), even after minimal monolayer expansion, lose osteogenic capacity in vivo. Communication between endothelial and stromal/mesenchymal cell lineages has been suggested to improve bone formation and vascularization by engineered tissues. Here, we investigated the specific role of a subpopulation of SVF cells positive for T-cadherin (T-cad), a putative endothelial marker. We found that maintenance during monolayer expansion of a T-cad-positive cell population, composed of endothelial lineage cells (ECs), is mandatory to preserve the osteogenic capacity of SVF cells in vivo and strongly supports their vasculogenic properties. Depletion of T-cad-positive cells from the SVF totally impaired bone formation in vivo and strongly reduced vascularization by SVF cells in association with decreased VEGF and Adiponectin expression. The osteogenic potential of T-cad-depleted SVF cells was fully rescued by co-culture with ECs from a human umbilical vein (HUVECs), constitutively expressing T-cad. Ectopic expression of T-cad in ASCs stimulated mineralization in vitro but failed to rescue osteogenic potential in vivo, indicating that the endothelial nature of the T-cad-positive cells is the key factor for induction of osteogenesis in engineered grafts based on SVF cells. This study demonstrates that crosstalk between stromal and T-cad expressing endothelial cells within adipose tissue critically regulates osteogenesis, with VEGF and adiponectin as associated molecular mediators.


Assuntos
Células Endoteliais , Osteogênese , Adiponectina/metabolismo , Tecido Adiposo , Caderinas , Diferenciação Celular , Células Cultivadas , Humanos , Células Estromais/metabolismo , Fração Vascular Estromal , Linfócitos T , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Cartilage ; 13(1): 19476035221075951, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35189712

RESUMO

OBJECTIVE: Implantation of tissue-engineered tracheal grafts represents a visionary strategy for the reconstruction of tracheal wall defects after resections and may develop into a last chance for a number of patients with severe cicatricial stenosis. The use of a decellularized tracheal substrate would offer an ideally stiff graft, but the matrix density would challenge efficient remodeling into a living cartilage. In this study, we hypothesized that the pores of decellularized laser-perforated tracheal cartilage (LPTC) tissues can be colonized by adult nasal chondrocytes (NCs) to produce new cartilage tissue suitable for the repair of tracheal defects. DESIGN: Human, native tracheal specimens, isolated from cadaveric donors, were exposed to decellularized and laser engraving-controlled superficial perforation (300 µm depth). Human or rabbit NCs were cultured on the LPTCs for 1 week. The resulting revitalized tissues were implanted ectopically in nude mice or orthotopically in tracheal wall defects in rabbits. Tissues were assayed histologically and by microtomography analyses before and after implantation. RESULTS: NCs were able to efficiently colonize the pores of the LPTCs. The extent of colonization (i.e., percentage of viable cells spanning >300 µm of tissue depth), cell morphology, and cartilage matrix deposition improved once the revitalized constructs were implanted ectopically in nude mice. LPTCs could be successfully grafted onto the tracheal wall of rabbits without any evidence of dislocation or tracheal stenosis, 8 weeks after implantation. Rabbit NCs, within the LPTCs, actively produced new cartilage matrix. CONCLUSION: Implantation of NC-revitalized LPTCs represents a feasible strategy for the repair of tracheal wall defects.


Assuntos
Gravuras e Gravação , Engenharia Tecidual , Animais , Cartilagem/transplante , Humanos , Lasers , Camundongos , Camundongos Nus , Coelhos , Engenharia Tecidual/métodos , Alicerces Teciduais
10.
Adv Mater ; 33(43): e2103737, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34486186

RESUMO

Design criteria for tissue-engineered materials in regenerative medicine include robust biological effectiveness, off-the-shelf availability, and scalable manufacturing under standardized conditions. For bone repair, existing strategies rely on primary autologous cells, associated with unpredictable performance, limited availability and complex logistic. Here, a conceptual shift based on the manufacturing of devitalized human hypertrophic cartilage (HyC), as cell-free material inducing bone formation by recapitulating the developmental process of endochondral ossification, is reported. The strategy relies on a customized human mesenchymal line expressing bone morphogenetic protein-2 (BMP-2), critically required for robust chondrogenesis and concomitant extracellular matrix (ECM) enrichment. Following apoptosis-driven devitalization, lyophilization, and storage, the resulting off-the-shelf cartilage tissue exhibits unprecedented osteoinductive properties, unmatched by synthetic delivery of BMP-2 or by living engineered grafts. Scalability and pre-clinical efficacy are demonstrated by bioreactor-based production and subsequent orthotopic assessment. The findings exemplify the broader paradigm of programming human cell lines as biological factory units to engineer customized ECMs, designed to activate specific regenerative processes.


Assuntos
Osteogênese
11.
iScience ; 19: 504-513, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31442666

RESUMO

The generation of humanized ectopic ossicles (hOss) in mice has been proposed as an advanced translational and fundamental model to study the human hematopoietic system. The approach relies on the presence of human bone marrow-derived mesenchymal stromal cells (hMSCs) supporting the engraftment of transplanted human hematopoietic stem and progenitor cells (HSPCs). However, the functional distribution of hMSCs within the humanized microenvironment remains to be investigated. Here, we combined genetic tools and quantitative confocal microscopy to engineer and subsequently analyze hMSCs' fate and distribution in hOss. Implanted hMSCs reconstituted a humanized environment including osteocytes, osteoblasts, adipocytes, and stromal cells associated with vessels. By imaging full hOss, we identified rare physical interactions between hMSCs and human CD45+/CD34+/CD90+ cells, supporting a functional contact-triggered regulatory role of hMSCs. Our study highlights the importance of compiling quantitative information from humanized organs, to decode the interactions between the hematopoietic and the stromal compartments.

12.
Acta Biomater ; 77: 142-154, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30126590

RESUMO

Many steps are required to generate bone through endochondral ossification with adipose mesenchymal stromal cells (ASC), from cell isolation to in vitro monolayer expansion, seeding into scaffolds, cartilaginous differentiation and in vivo remodeling. Moreover, monolayer expansion and passaging of ASC strongly decreases their differentiation potential. Here, we propose that adipose tissue itself can be used as scaffold for ASC expansion and endochondral ossification. Human liposuctions were fractionated and cultured for 3 weeks with proliferative medium in suspension. The resulting constructs, named Adiscaf, were compared to constructs generated with a previously developed, control approach, i.e. collagen sponges seeded with monolayer-expanded ASC. After 4 weeks of chondrogenic differentiation, Adiscaf contained cartilage tissue, characterized by glycosaminoglycans and collagen type II. After 2 additional weeks of hypertrophic differentiation, Adiscaf showed upregulation of hypertrophic markers at the gene expression and protein levels. After 8 weeks of in vivo implantation, Adiscaf resulted in ectopic bone tissue formation, including bone marrow elements. Adiscaf showed superior in vitro differentiation and in vivo performance as compared to the control paradigm involving isolation and monolayer expansion of ASC. This new paradigm exploits the physiological niche of adipose tissue and strongly suggests a higher functionality of cells inside adipose tissue after in vitro expansion. This study demonstrates that adult human adipose tissue used as a native construct can generate a bone organ by endochondral ossification. The concept could be exploited for the generation of osteogenic grafts for bone repair. STATEMENT OF SIGNIFICANCE: In this study we used adult human adipose tissue as scaffolding materials (called Adiscaf) to generate a bone organ by endochondral ossification. Adiscaf concept is based on the culture of adipose tissue cells inside their native microenvironment for the generation of osteogenic grafts for bone repair. This simplified approach overcomes several limitations linked to the current techniques in bone tissue engineering, such as isolation of cells and inadequate properties of the biomaterials used as scaffolds. In addition, the present paradigm proposes to exploit physiological niches in order to better maintain the functionality of cells during their in vitro expansion. This project not only has a scientific impact by evaluating the impact of native physiological niches on the functionality and chondrogenic differentiation of mesenchymal progenitors but also a clinical impact to generate osteogenic grafts and/or osteoinductive materials for bone regeneration and repair.


Assuntos
Tecido Adiposo/citologia , Materiais Biocompatíveis/química , Regeneração Óssea , Células-Tronco Mesenquimais/citologia , Osteogênese , Idoso , Animais , Substitutos Ósseos , Transplante Ósseo , Osso e Ossos/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/citologia , Condrogênese/genética , Colágeno/química , Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Lipectomia , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Microtomografia por Raio-X
13.
Exp Hematol ; 61: 45-51.e5, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29410245

RESUMO

Hematopoietic stem cells (HSCs) are maintained in a specialized bone marrow (BM) environment, the so-called HSC niche, that provides pivotal factors for their maintenance. Although the cellular and molecular components of the mouse BM HSC niche have been extensively studied using genetically modified animals, relatively little is known about the counterpart human BM niche components. We previously illustrated, with a developmental tissue engineering approach, that human adult BM-derived mesenchymal stromal cells (MSCs) can develop into human bone organs (so-called ossicles) through endochondral ossification in vivo and that these human ossicles are able to maintain functional mouse HSCs. We here report that human ossicles in immunodeficient mice maintain human immature and mature hematopoiesis in vivo. Moreover, a higher percentage of human stem and progenitor cells are kept in quiescence in human ossicles as compared with mouse BM. These findings indicate that the human MSC-derived ossicles function as a hematopoietic niche and may potentially serve as a re-engineerable platform to study normal and diseased human hematopoiesis in a physiologically optimized environment.


Assuntos
Materiais Biocompatíveis/metabolismo , Células da Medula Óssea/citologia , Osso e Ossos/citologia , Hematopoese/fisiologia , Animais , Bioengenharia , Humanos , Camundongos , Nicho de Células-Tronco , Transplante de Células-Tronco
14.
Stem Cell Reports ; 11(2): 440-453, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30057264

RESUMO

Cartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. When transplanted subcutaneously, most of the surviving pellets were fully mineralized by 8 weeks. In contrast, treatment with the adenylyl cyclase activator, forskolin, in vitro resulted in slightly enlarged cartilage pellets containing an increased proportion of proliferating immature chondrocytes that expressed very low levels of hypertrophic/terminally matured chondrocyte-specific genes. Forskolin treatment also enhanced hyaline cartilage formation by reducing type I collagen gene expression and increasing sulfated glycosaminoglycan accumulation in the developed cartilage. Chondrogenic mesoderm from hPSCs and dedifferentiated nasal chondrocytes responded similarly to forskolin. Furthermore, forskolin treatment in vitro increased the frequency at which the cartilage pellets maintained unmineralized chondrocytes after subcutaneous transplantation. Thus, the post-transplantational fate of chondrocytes originating from hPSC-derived chondroprogenitors can be controlled during their genesis in vitro.


Assuntos
Cartilagem/citologia , Diferenciação Celular , Condrócitos/citologia , Condrogênese , Células-Tronco Pluripotentes/citologia , Biomarcadores , Proteína Morfogenética Óssea 4/metabolismo , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Colforsina/farmacologia , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo XI/genética , Colágeno Tipo XI/metabolismo , Biologia Computacional/métodos , AMP Cíclico/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Ontologia Genética , Glicosaminoglicanos/biossíntese , Humanos , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco
15.
Stem Cell Res ; 12(2): 584-98, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24561906

RESUMO

The hTERT-immortalization of human bone marrow-derived Mesenchymal Stromal Cells (hMSCs) was proposed to address availability/standardization issues for experimental or clinical studies, but raised concerns due to possible uncontrolled growth or malignant cell transformation. Here we report a method to generate a hMSCs line with controlled survival, through the implementation of a pre-established suicide system (inducible caspase 9, iCasp9) in hTERT-transduced hMSCs. Primary hMSCs were successfully immortalized (>280 PD) and further transduced with the iCasp9 device. A clone was selected and shown to maintain typical properties of primary hMSCs, including phenotype, differentiation and immunomodulation capacities. The successive transductions did not induce tumorigenic transformation, as assessed by analysis of cell cycle regulators and in vivo luciferase-based cell tracking. Cells could be efficiently induced toward apoptosis (>95%) both in vitro and in vivo. By combining the opposite concepts of 'induced-life' and 'inducible-death', we generated a hMSCs line with defined properties and allowing for temporally controlled survival. The cell line represents a relevant tool for medical discovery in regenerative medicine and a potential means to address availability, standardization and safety requirements in cell & gene therapy. The concept of a hTERT-iCasp9 combination, here explored in the context of hMSCs, could be extended to other types of progenitor/stem cells.


Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Animais , Células da Medula Óssea/metabolismo , Morte Celular/fisiologia , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Feminino , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade
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