Patient Adipose Stem Cell-Derived Adipocytes Reveal Genetic Variation that Predicts Antidiabetic Drug Response

Hu, Wenxiang; Jiang, Chunjie; Guan, Dongyin; Dierickx, Pieterjan; Zhang, Rong; Moscati, Arden; Nadkarni, Girish N.; Steger, David J.; Loos, Ruth J.F.; Hu, Cheng; Jia, Weiping; Soccio, Raymond E.; Lazar, Mitchell A.

doi:10.1016/j.stem.2018.11.018

Cited by 27 publications

(23 citation statements)

References 40 publications

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“…Most importantly, in cultured primary adipocytes derived from 26 different individuals, we show that this variant predicts transcriptional response of PM20D1 to TZD drugs. We used the same approach in our recent report showing another variant driving TZD regulation of ABCA1 (28). Such examples confirm that natural noncoding genetic variation in nuclear receptor genomic binding sites can determine differences in transcriptional response to drugs (27,29).…”

Section: Discussionmentioning

confidence: 72%

“…Mouse 3T3-L1 adipocytes and human SGBS adipocytes were differentiated as previously described (14). Primary human adipose stem cellderived adipocytes were differentiated from the stromal vascular fraction of fat biopsies and treated with rosiglitazone, as previously described (28). Note that both SGBS cells and primary adipocytes were generated in the presence of rosiglitazone, as TZD is required for differentiation, but they were cultured in maintenance media lacking this drug for 11 or 7 d prior to the rosiglitazone treatment at 1 μM for 24 to 48 h. 293T cells (ATCC) were maintained at 37°C in 5% CO 2 in high glucose DMEM supplemented with 10% fetal bovine serium and L-glutamine (Gibco).…”

Section: Methodsmentioning

confidence: 99%

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Natural human genetic variation determines basal and inducible expression of PM20D1 , an obesity-associated gene

Benson

Weller

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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PM20D1 is a candidate thermogenic enzyme in mouse fat, with its expression cold-induced and enriched in brown versus white adipocytes. Thiazolidinedione (TZD) antidiabetic drugs, which activate the peroxisome proliferator-activated receptor-γ (PPARγ) nuclear receptor, are potent stimuli for adipocyte browning yet fail to induce Pm20d1 expression in mouse adipocytes. In contrast, PM20D1 is one of the most strongly TZD-induced transcripts in human adipocytes, although not in cells from all individuals. Two putative PPARγ binding sites exist near the gene’s transcription start site (TSS) in human but not mouse adipocytes. The −4 kb upstream site falls in a segmental duplication of a nearly identical intronic region +2.5 kb downstream of the TSS, and this duplication occurred in the primate lineage and not in other mammals, like mice. PPARγ binding and gene activation occur via this upstream duplicated site, thus explaining the species difference. Furthermore, this functional upstream PPARγ site exhibits genetic variation among people, with 1 SNP allele disrupting a PPAR response element and giving less activation by PPARγ and TZDs. In addition to this upstream variant that determines PPARγ regulation of PM20D1 in adipocytes, distinct variants downstream of the TSS have strong effects on PM20D1 expression in human fat as well as other tissues. A haplotype of 7 tightly linked downstream SNP alleles is associated with very low PMD201 expression and correspondingly high DNA methylation at the TSS. These PM20D1 low-expression variants may account for human genetic associations in this region with obesity as well as neurodegenerative diseases.

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Section: Discussionmentioning

confidence: 72%

Section: Methodsmentioning

confidence: 99%

Natural human genetic variation determines basal and inducible expression of PM20D1 , an obesity-associated gene

Benson

Weller

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Genome editing was so far mainly done in immortalized or transformed cell lines [31] and an overall efficiency up to ~80% has been reported [17]. For instance, Hu et al used the human SGBS preadipocyte cell line for CRISPR/Cas9-mediated gene correction due to its higher susceptibility to genome editing compared to patient-derived ASCs [32]. However, performing such experiments in primary human cells, such as ASCs, is a technical challenge [31].…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells

et al. 2020

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The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/ Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types.

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“…In addition, the derivation of these cell types from hiPSC or primary progenitors can aid in understanding patient- and disease-specific responses to drugs, unlike models constructed of cell lines. For example, the use of in vitro adipose tissues derived from various patients' adipose stromal cells revealed genetic variations that predict the variable patient response to the thiazolidinedione PPARγ agonist, Rosiglitazone [ 172 ]. Additionally, recent studies have demonstrated the capacity for more precise specification of the phenotype of hPSC-derived cell types.…”

Section: Biological Advances: Improving Access To Disease-relevant Humentioning

confidence: 99%

Engineered tissues and strategies to overcome challenges in drug development

Khalil

Jaenisch

Mooney

2020

Advanced Drug Delivery Reviews

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Current preclinical studies in drug development utilize high-throughput in vitro screens to identify lead drug candidates, followed by both in vitro and in vivo models to predict lead candidates' pharmacokinetic and pharmacodynamic properties. The goal of these studies is to reduce the number of lead drug candidates down to the most likely to succeed in later human clinical trials. However, only 1 in 10 drug candidates that emerge from preclinical studies will succeed and become an approved therapeutic. Lack of efficacy or undetected toxicity represents roughly 75% of the causes for these failures, despite these parameters being the primary exclusion criteria in preclinical studies. Recently, advances in both biology and engineering have created new tools for constructing new preclinical models. These models can complement those used in current preclinical studies by helping to create more realistic representations of human tissues in vitro and in vivo . In this review, we describe current preclinical models to identify their value and limitations and then discuss select areas of research where improvements in preclinical models are particularly needed to advance drug development. Following this, we discuss design considerations for constructing preclinical models and then highlight recent advances in these efforts. Taken together, we aim to review the advances as of 2020 surrounding the prospect of biological and engineering tools for adding enhanced biological relevance to preclinical studies to aid in the challenges of failed drug candidates and the burden this poses on the drug development enterprise and thus healthcare.

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Patient Adipose Stem Cell-Derived Adipocytes Reveal Genetic Variation that Predicts Antidiabetic Drug Response

Cited by 27 publications

References 40 publications

Natural human genetic variation determines basal and inducible expression of PM20D1 , an obesity-associated gene

Natural human genetic variation determines basal and inducible expression of PM20D1 , an obesity-associated gene

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells

Engineered tissues and strategies to overcome challenges in drug development

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