Estudos moleculares de células tronco mesenquimais cultivadas in vitro

Células Tronco Mesenquimais (CTM) podem ser expandidas ex vivo e são capazes de se diferenciar em diversas linhagens, incluindo condrócitos, osteoblastos e adipócitos. Elas secretam uma série de citocinas e moléculas regulatórias implicadas em diferentes aspectos da hematopoese, e parecem modular o sistema imune. Devido a essas características, as CTM representam promissoras ferramentas para a terapia celular, sobretudo aquelas associadas ao transplante de medula óssea. O objetivo deste trabalho foi melhorar a caracterização molecular de culturas de CTM, através da utilização de ferramentas genômicas e proteômicas. O perfil de expressão proteica de culturas de CTM foi analisado através de ensaios de eletroforese bidimensional e espectrometria de massas in tandem. Na janela de análise utilizada (pH 4-7, PM 10-220 KDa), foram identificadas 84 proteínas distintas em culturas de CTM. Nossas análises mostraram um padrão proteômico muito similar entre culturas de CTM derivadas de diferentes doadores já na primeira passagem, sugerindo que as células apresentam o mesmo padrão de expressão protéica. Além disso, culturas derivadas de diferentes doadores são igualmente capazes de inibir a proliferação linfocitária in vitro. A análise comparativa dos padrões genômicos e proteômicos de CTM submetidas a diferentes passagens em cultura, mostrou que o procedimento de cultivo in vitro de células estromais é capaz de uniformizar a população em cultura, pelo menos no que diz respeito a seu perfil molecular. Apesar de manterem o potencial imunomodulatório in vitro, as células adquiriram, depois da passagem 5, alterações no seu cariótipo, indicando que elas apresentam instabilidade genética em cultura. Análises proteômicas de CTM submetidas à diferenciação adipogênica e osteogênica sugerem ainda que a diferenciação in vitro das CTM altera de forma importante o perfil molecular das culturas, afetando a expressão de diversas proteínas, como as envolvidas na estrutura do esqueleto de actina das células.

Mesenchymal Stem Cells (MSC) can be expanded ex vivo and are able to differentiate along multiple lineages, including chondrocytes, osteoblasts and adipocytes. They secrete a number of cytokines and regulatory molecules implicated in different aspects of hematopoiesis, and seem to modulate the immune system. Because of these characteristics, MSC represent a promissing tool for cellular therapies, especially that related to bone marrow transplantation. The aim of this work was to improve the molecular characterization of MSC cultures using genomic and proteomic tools. Protein expression profile was analyzed by bidimensional electrophoresis and in tandem mass spectrometry. In a window of observation (pH 4-7, MW 10-220 KDa), 84 distinct proteins were identified in MSC cultures. Our analyses demonstrated a very similar proteomic profile of MSC cultures of the first passage derived from different donors, suggesting that these cells have the same expression pattern. Additionally, cells derived from different donors were able to equally inhibit lymphocyte proliferation. Comparative analyses of genomic and proteomic pattern of MSC under different culture passages showed that the in vitro cultivation procedure is able to standardize the cells, in terms of their molecular profile. Although MSC maintain their in vitro immunomodulatory potential, after passage 5 they developed karyotypic alterations, indicating genetic instability of cultured cells. Proteomic analyses of MSC submitted to adipogenic and osteogenic differentiation suggest that these processes strongly modify their molecular profile, affecting the expression of various proteins, such as those involved in actin skeleton of cells

Possible role of Msx1 in murine hematopoiesis

The Msx1 gene is expressed at sites of epitelium-mesenchymal interaction throughout development and is important in morphogenesis since Msx1 null mice die 24 h. after birth and show defects in craniofacial bones. The Msx1 gene code for a transcription factor activated by BMP4 and, when active, maintains progenitor cells in an undifferentiated state. BMP4 is a crucial factor for hematopoietic development in the murine embryo and although Msx1 is activated by BMP4, there have been no reports relating Msx1 to hematopoiesis. To investigate the role of Msx1 in murine hematopoiesis, samples of hematopoietic tissues (spleen, liver, thymus, bone marrow) and blood collected from 18.5 days post coitum (dpc) fetuses of Msx1 mutant and wild-type embryos were analyzed histologically. Blood cell counts as well as erythropoietic clonogenic assays for liver and bone marrow cells were also done. Histological analysis of the spleen suggested the presence of fewer erythrocytes but more hematopoietic progenitors in mutant embryos. While the bone marrow of wild-type mice had mesenchymal and hematopoietic components, in mutant mice only the hematopoietic component was seen. Hematopoietic progenitors and more mature cells, as well as extracellular matrix, filled the entire bone marrow in heterozygous mutants. In homozygous null mice, this phenotype was enhanced with a poor bone marrow. In hematopoietic colony assays, the liver and bone marrow of Msx1 knockout embryos had a higher number of erythropoietic progenitors whereas peripheral blood had a lower number of erythrocytes compared to wild-type mice. These results suggest that Msx1 plays a role in erythropoietic differentiation since low or null gene expression increased the level of progenitors and decreased the number of differentiated erythroid cells. Msx1 appears to act in mesenchymal cells since in Msx1-/-embryos the hematopoietic cells were abnormal and the cells that supported hematopoietic development in bone marrow were missing.