RESUMEN
Human adipose derived stem cells (hASCs) can be easily isolated and their plasticity has been well characterized. Several TGF-ß superfamily ligands can direct hASCs towards chondrocytes. However, these ligands are difficult to purify and expensive. We have developed a library of Activin/BMP2 chimeric ligands (AB2 ligands) by systematically mixing their sequence segments and have tested their chondrogenic potential in hASCs. Cells cultured in monolayer or in a pellet culture system were incubated with a chemically defined medium supplemented with the chimeric ligands for 4 or 6 weeks and showed higher expression levels of type II collagen, aggrecan, and Sox9 mRNAs when compared with control and non-treated cells. Moreover, toluidine blue, alcian blue, and Masson's trichrome staining was markedly increased in treated cells, both in cell pellet and monolayer assays. In addition, immunohistochemical staining for detection of type I collagen, type II collagen, and Sox 9 demonstrated the acquisition of a chondrogenic phenotype in both culture systems. We present here an inexpensive and robust protocol for differentiation of hASCs towards chondrocytes in a reproducible and highly efficient manner. The AB2 ligands employed are easily produced and have properties that may become useful in cell therapy.
Asunto(s)
Tejido Adiposo/citología , Condrocitos/citología , Células Madre Mesenquimatosas/citología , Activinas/genética , Activinas/metabolismo , Adulto , Agrecanos/genética , Agrecanos/metabolismo , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/metabolismo , Condrogénesis/efectos de los fármacos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Femenino , Humanos , Ligandos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
Articular cartilage, which is mainly composed of collagen II, enables smooth skeletal movement. Degeneration of collagen II can be caused by various events, such as injury, but degeneration especially increases over the course of normal aging. Unfortunately, the body does not fully repair itself from this type of degeneration, resulting in impaired movement. Microfracture, an articular cartilage repair surgical technique, has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However, the therapeutic outcomes of all these techniques vary in different patients depending on their age, health, lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage, both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone, or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs), which are able to self-renew and differentiate into multiple cell types, provides a potentially valuable cell resource for drug screening in a "more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis.
Asunto(s)
Condrogénesis/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Diferenciación Celular/efectos de los fármacos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Genes Reporteros/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Luciferasas/genética , Péptidos/síntesis química , Péptidos/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs, progerin and its ageing-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs, also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing.
