Ultrastructural analysis of different human mesenchymal stem cells after in vitro expansion: a technical review

Abstract

Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissue-derived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc.) and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature) while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering.

Ultraestructura del Inmunofenotipo CD44+/CD24- de una Línea de Células de Cáncer de Mama Triple Negativo

“…Con el propósito de tener un patrón morfológico de células madre normales, estudiamos células madre adiposas de pasaje 2-5 aisladas y caracterizadas en nuestro laboratorio, las que son de tamaño heterogéneo y crecen en monocapas formando colonias separadas por distancias cortas. Similar a lo que describen otros autores, las células estudiadas en este trabajo, tienen numerosas gotas de grasa en el citoplasma, en el núcleo tienen varios nucléolos y están recubiertas por abundantes microvellosidades largas y adheridas al sustrato mediante filipodios (Miko et al, 2015), fenotipo compatible a una fase de síntesis proteica.…”

Histochemistry today: Detection and location of single molecules

“…Several papers were aimed at illustrating the tissue organization and protein expression of still poorly described organs from various Vertebrate and Invertebrate species: [42][43][44][45][46][47][48][49][50][51][52][53][54] here, the morphological description of the microanatomical and histological features was paralleled by the detection of specific molecules responsible for the tissue functional characteristics, also as a consequence of seasonal dynamic changes. [55][56][57] The immunolabeling of marker proteins was extensively applied in studies aimed at distinguishing stem/progenitor cells or at elucidating the process of cell differentiation and morphogenesis during pre-and post-natal development in mammals, including humans, [58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76] with special attention to the nervous system, 65,66 sensory organs, [67][68][69] skin, 70 lung, 70,71 and the skeletal apparatus. [72][73][74][75] Also in plants, the histochemical d...…”

Is there still room for novelty, in histochemical papers?

“…Viable chondrogenic stem cells have been detected in the knee joint loose body, 71 and the ultrastructural features of mesenchymal stem cells after in vitro expansion have been reviewed. 72 Histochemistry proved to be especially suitable also for the essentially descriptive articles on Tissue Biology in Human & Animals. 73-98 Here, several proteins and protein receptors have been studied in different organs and tissues from mammals (rodents, 73-82 bat, 83 dog, 84 primates and human 85-88 ), as well as from lower vertebrates 89-94 and invertebrates.…”