Understanding Human Lung Development through In Vitro Model Systems.

An abundance of information about lung development in animal models exists; however, comparatively little is known about lung development in humans. Recent advances using primary human lung tissue combined with the use of human in vitro model systems, such as human pluripotent stem cell-derived tissue, have led to a growing understanding of the mechanisms governing human lung development. They have illuminated key differences between animal models and humans, underscoring the need for continued advancements in modeling human lung development and utilizing human tissue. This review discusses the use of human tissue and the use of human in vitro model systems that have been leveraged to better understand key regulators of human lung development and that have identified uniquely human features of development. This review also examines the implementation and challenges of human model systems and discusses how they can be applied to address knowledge gaps.

In Vitro and In Vivo Development of the Human Airway at Single-Cell Resolution.

Bud tip progenitor cells give rise to all murine lung epithelial lineages and have been described in the developing human lung; however, the mechanisms controlling human bud tip differentiation into specific lineages are unclear. Here, we used homogeneous human bud tip organoid cultures and identified SMAD signaling as a key regulator of the bud tip-to-airway transition. SMAD induction led to the differentiation of airway-like organoids possessing functional basal cells capable of clonal expansion and multilineage differentiation. To benchmark in vitro-derived organoids, we developed a single-cell mRNA sequencing atlas of the human lung from 11.5 to 21 weeks of development, which revealed high degrees of similarity between the in vitro-derived and in vivo airway. Together, this work sheds light on human airway differentiation in vitro and provides a single-cell atlas of the developing human lung.

A Purification Technique for Adipose-Derived Stromal Cell Cultures Leads to a More Regenerative Cell Population.

It has been demonstrated that adipose-derived stromal cells (ASCs) are a regenerative cell population with potential uses for bone and cartilage regeneration. However, the biomarker expression and heterogeneity of the population has not been thoroughly characterized. By analyzing biomarker expression, we aimed to understand the composition of ASC populations extracted using a common extraction technique in comparison to ASC populations given an additional purification step. Human adipose tissue samples were collected, and ASCs were extracted from these samples using a common, published extraction technique (primary extraction). These cells were cultured and half were given an additional purification. The primarily-extracted and purified cell populations were analyzed for biomarkers that correspond to specific cell types. The addition of the purification technique reduced the number of cells expressing hematopoietic and endothelial biomarkers and did not cause the yield of mesenchymal stem cell biomarker-expressing cells to decrease. Biomarkers corresponding to erythrocytes and lymphocytes were lost during the primary extraction, and biomarkers corresponding to most granulocytes and progenitor cells were lost during the additional purification. Biomarkers identifying dendritic cells, monocytes/macrophages, neutrophils, vascular endothelial cells, smooth muscle cells, and pericytes were upregulated in purified cell populations while those identifying fibroblasts and adipocytes were downregulated. Pluripotency biomarkers were more highly expressed in purified cell populations. These results demonstrate that the most commonly utilized adipose tissue recovery and ASC extraction technique leads to a heterogeneous cell population in which further purification of this population, as described in this manuscript, isolates a cell subset that has more regenerative potential.

Adipose-derived human stem/stromal cells: comparative organ specific mitochondrial bioenergy profiles.

BACKGROUND: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen. RESULTS: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive for MSC markers CD90, CD73, and CD105, and negative for hematopoietic markers CD34, CD11B, CD19, and CD45. Bioenergetic profiles were obtained for ASCs with an n = 4 for each tissue type and graphed together for comparison. Mitochondrial stress tests provided the following measurements: basal respiration rate (measured as oxygen consumption rate [pmol O2/min], ATP production, proton leak, maximal respiration, respiratory control ratio, coupling efficiency, and non-mitochondrial respiration. Glycolytic stress tests provided the following measurements: basal glycolysis rate (measured as extracellular acidification rate [mpH/min]), glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. CONCLUSIONS: The main goal of this manuscript was to provide baseline reference values for future experiments and to compare bioenergetic potentials of ASCs isolated from adipose tissue harvested from different anatomical locations. Through an investigation of mitochondrial respiration and glycolysis, it was demonstrated that bioenergetic profiles do not significantly differ by region due to depot-dependent and donor-dependent variability. Thus, although the physiological function, microenvironment and anatomical harvest site may directly affect the characteristics of ASCs isolated from different organ regions, the ultimate utility of ASCs remains independent of the anatomical harvest site.