Subcutaneous Leydig Stem Cell Autograft: A Promising Strategy to Increase Serum Testosterone.

Exogenous testosterone therapy can be used to treat testosterone deficiency; however, it has several adverse effects including infertility due to negative feedback on the hypothalamic-pituitary-gonadal (HPG) axis. Leydig stem cell (LSC) transplantation could provide a new strategy for treating testosterone deficiency, but clinical translatability of injecting stem cells inside the testis is not feasible. Here, we explore the feasibility of subcutaneously autografting LSCs in combination with Sertoli and myoid cells to increase testosterone. We also studied whether the grafted LSCs can be regulated by the HPG axis and the molecular mechanism behind this regulation. LSCs were isolated from the testes of 12-week-old C57BL/6 mice, and subcutaneously autografted in combination with Sertoli cells and myoid cells. We found that LSCs alone were incapable of self-renewal and differentiation. However, in combination with Sertoli cells and myoid cells, LSCs underwent self-renewal as well as differentiation into mature Leydig cells. As a result, the recipient mice that received the LSC autograft showed testosterone production with preserved luteinizing hormone. We found that testosterone production from the autograft was regulated by hedgehog (HH) signaling. Gain of function and loss of function study confirmed that Desert HH (DHH) agonist increased and DHH antagonist decreased testosterone production from autograft. This study is the first to demonstrate that LSCs, when autografted subcutaneously in combination with Sertoli cells and myoid cells, can increase testosterone production. Therefore, LSC autograft may provide a new treatment for testosterone deficiency while simultaneously preserving the HPG axis. Stem Cells Translational Medicine 2019;8:58-65.

In vitro heterogeneity of porcine adipose tissue-derived stem cells

Tissue-specific adult stem cells (ASC) are heterogeneous and characterized by a mix of progenitor cells that produce cells at various stages of differentiation, and ultimately different terminally differentiated cells. Understanding the heterogeneity of ASCs may lead to the development of improved protocols of cell isolation and optimized cell therapy clinical protocols. Using a combination of enzymatic and explant culture protocols, we obtained pADSC population, which is composed by two distinct morphologies: fibroblast-like cells (FLCs) and endothelial-like cells (ELCs). Both cell sub-types efficiently formed colonies, expressed CD90+/CD105+/CD44+, and differentially expressed such markers such as Nestin, Vimentin, Fibronectin, Cytokeratin, Connexin 43, CD31, CD34 and CD146 as well as the pluripotent stem cell markers Oct-4, Nanog and Sox2. Mixed populations of pADSCs did not lose their multipotentiality and the cells were able to undergo osteogenic, chondrogenic, adipogenic and myogenic differentiation. Furthermore, the mixed population spontaneously formed capillary tube structures. Our findings suggest that different subpopulations can be isolated from adipose tissue and that the ADSCs need to be better evaluated using a wide panel of different markers related to cell differentiation, which is important for stem cell therapy and regenerative medicine, particularly for advanced stem cells therapies – products that are currently under investigation or even use.

In vitro heterogeneity of porcine adipose tissue-derived stem cells

Tissue-specific adult stem cells (ASC) are heterogeneous and characterized by a mix of progenitor cells that produce cells at various stages of differentiation, and ultimately different terminally differentiated cells. Understanding the heterogeneity of ASCs may lead to the development of improved protocols of cell isolation and optimized cell therapy clinical protocols. Using a combination of enzymatic and explant culture protocols, we obtained pADSC population, which is composed by two distinct morphologies: fibroblast-like cells (FLCs) and endothelial-like cells (ELCs). Both cell sub-types efficiently formed colonies, expressed CD90+/CD105+/CD44+, and differentially expressed such markers such as Nestin, Vimentin, Fibronectin, Cytokeratin, Connexin 43, CD31, CD34 and CD146 as well as the pluripotent stem cell markers Oct-4, Nanog and Sox2. Mixed populations of pADSCs did not lose their multipotentiality and the cells were able to undergo osteogenic, chondrogenic, adipogenic and myogenic differentiation. Furthermore, the mixed population spontaneously formed capillary tube structures. Our findings suggest that different subpopulations can be isolated from adipose tissue and that the ADSCs need to be better evaluated using a wide panel of different markers related to cell differentiation, which is important for stem cell therapy and regenerative medicine, particularly for advanced stem cells therapies – products that are currently under investigation or even use.

Células-tronco de tecido adiposo e a importância da padronização de um modelo animal para experimentação pré-clínica / Adipose tissue-derived stem cells and the importance of animal model standardization for pre-clinical trials

As células-tronco são células indiferenciadas, capazes de se autorrenovar e de se diferenciarem em diversos tipos celulares, além de apresentarem propriedades imunomoduladoras e efeitos parácrinos mediante injúria tecidual, podendo, dessa forma, tratar lesões e doenças ou ainda substituir células danificadas ou perdidas. Dentre as fontes de células-tronco adultas, o tecido adiposo é uma fonte atrativa, pois o organismo humano possui grande reserva desse tecido, que, por sua vez, é obtido em grandes quantidades por meio de métodos pouco invasivos. O interesse nessas células vem aumentando constantemente devido a suas propriedades e possíveis aplicações na medicina regenerativa e terapia celular. Grande parte dessas pesquisas está voltada para doenças cardiovasculares, que são a principal causa de morbidade e mortalidade em todo o mundo. Embora nos últimos anos, os tratamentos em cardiologia tenham avançado, o desenvolvimento de novas terapias que recuperem o tecido danificado ainda permanece como um dos objetivos principais das pesquisas cardíacas. Porém, para obter resultados eficazes, é necessária a padronização de modelos animais in vivo e in vitro para estudos pré-clínicos e, consequentemente, a aplicação em humanos. O desenvolvimento de modelos pré-clínicos em animais de grande porte exige o uso bem caracterizado de linhagens de células animais semelhantes aos seus equivalentes humanos. O modelo suíno representa uma grande vantagem para a investigação translacional pré-clínica.

Stem cells are undifferentiated cells and can self-renew and differentiate into various cell types, besides having immunomodulating properties and paracrine effects in response to tissue injury, and may therefore treat injuries and diseases or even replace damaged or lost cells. Adipose tissue is an attractive source of adult stem cells, since the human body has a large reserve that is obtained in large amounts by minimally invasive methods. Interest in these cells has been increasing steadily due to their properties and possible applications in regenerative medicine and cell therapy. A large part of these investigations are focused on cardiovascular diseases, which are a leading cause of morbidity and mortality worldwide. Although in recent years treatments have advanced in cardiology, the development of new therapies to recover the damaged tissue still remains one of the main goals of cardiac research. However, to achieve effective results, in vivo and in vitro animal models for preclinical studies and consequently for application in humans must be standardized. The development of preclinical models in large animals requires the use of well-characterized animal cell lines, similar to human cells, and the use of the porcine model represents a great advantage for preclinical translational research.