Heat-producing beige/brite (brown-in-white) adipocytes in white adipose tissue have the potential to suppress metabolic disease in mice and hold great promise for the treatment of obesity and type 2 diabetes in humans. Here, we demonstrate that human adipose-derived stromal/progenitor cells (hASCs) from subcutaneous white adipose tissue can be efficiently converted into beige adipocytes. Upon pharmacological activation of peroxisome proliferator-activated receptor-γ, hASC-derived adipocytes activated beige fat-selective genes and a brown/beige fat-selective electron transport chain gene program. Importantly, hASC-derived beige fat cells displayed the bioenergetic characteristics of genuine brown fat cells, including a capacity for increased respiratory uncoupling in response to β-adrenergic agonists. Furthermore, knock-down experiments reveal that the thermogenic capacity of human beige fat cells was entirely dependent on the presence of Uncoupling protein 1. In summary, this study reveals that hASCs can be readily differentiated into beige adipocytes that, upon activation, undergo uncoupling protein 1-dependent thermogenesis.

The mechanisms regulating T lymphocyte migration within the extracellular matrix are not understood. We show in this study that the thrombospondin-1 binding site of calreticulin, spanning aa 19–32, is a major triggering factor for T cell motility and migration within a three-dimensional collagen type 1 matrix, and that exogenous motogenic factors such as chemokines can stimulate migration via a calreticulin-thrombospondin-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous thrombospondin-1 elicited a motogenic signal to the T cells through the C-terminal domain of thrombospondin-1 and its cell surface receptor integrin-associated protein (CD47). Our data further revealed that thrombospondin-1 was expressed on the cell surface with a high turnover, and that PI3K and the Janus family of tyrosine kinases were required for T cell motility mediated through calreticulin, thrombospondin-1, and CD47. These results unveil an autocrine mechanism of calreticulin-thrombospondin-1-CD47 interaction for the control of T cell motility and migration within three-dimensional extracellular matrix substrata.

Chemokines are key regulators of cell trafficking, and dipeptidyl peptidase IV/CD26 (CD26) inactivates chemokines. Here we show that the CD26-processed chemokines SDF1α/CXCL12 and RANTES/CCL5, in contrast to a control chemokine not processed by CD26, are potent inducers of cell surface expression of thrombospondin-1 (TSP-1) in T lymphocytes through a CD26-controlled mechanism and that TSP-1 stimulates expression of lipoprotein receptor related protein/CD91. Accordingly, intact TSP-1 and a peptide mimetic of a sequence in TSP-1 were sufficient to stimulate CD91 expression. The chemokine-induced expression of TSP-1 and CD91 was mimicked by inhibitors of CD26 and CXCL12 and CCL5 as well as inhibitors of CD26 stimulated polarized cytoplasmic spreading and migration through TSP-1. Silencing of CD26 using small interfering RNA or Ab-induced modulation of CD26 also increased TSP-1 expression and enhanced cytoplasmic spreading and T cell migration markedly. These results indicate that CD26 is an endogenous inhibitor of T cell motility through inhibition of TSP-1 expression and that chemokines stimulate cell polarity and migration through abrogation of the CD26-dependent inhibition. This suggests that T cell motility is regulated by a cascade of interacting cell surface molecules.

Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.