Dataset Information

Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation.

ABSTRACT:

Background

To maximize the translational utility of human induced pluripotent stem cells (iPSCs), the ability to precisely modulate the differentiation of iPSCs to target phenotypes is critical. Although the effects of the physical cell niche on stem cell differentiation are well documented, current approaches to direct step-wise differentiation of iPSCs have been typically limited to the optimization of soluble factors. In this regard, we investigated how temporally varied substrate stiffness affects the step-wise differentiation of iPSCs towards various lineages/phenotypes.

Methods

Electrospun nanofibrous substrates with different reduced Young's modulus were utilized to subject cells to different mechanical environments during the differentiation process towards representative phenotypes from each of three germ layer derivatives including motor neuron, pancreatic endoderm, and chondrocyte. Phenotype-specific markers of each lineage/stage were utilized to determine differentiation efficiency by reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence imaging for gene and protein expression analysis, respectively.

Results

The results presented in this proof-of-concept study are the first to systematically demonstrate the significant role of the temporally varied mechanical microenvironment on the differentiation of stem cells. Our results demonstrate that the process of differentiation from pluripotent cells to functional end-phenotypes is mechanoresponsive in a lineage- and differentiation stage-specific manner.

Conclusions

Lineage/developmental stage-dependent optimization of electrospun substrate stiffness provides a unique opportunity to enhance differentiation efficiency of iPSCs for their facilitated therapeutic applications.

SUBMITTER: Maldonado M

PROVIDER: S-EPMC5622562 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Publications

Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation.

Maldonado Maricela M Luu Rebeccah J RJ Ico Gerardo G Ospina Alex A Myung Danielle D Shih Hung Ping HP Nam Jin J

Stem cell research & therapy 20170929 1

<h4>Background</h4>To maximize the translational utility of human induced pluripotent stem cells (iPSCs), the ability to precisely modulate the differentiation of iPSCs to target phenotypes is critical. Although the effects of the physical cell niche on stem cell differentiation are well documented, current approaches to direct step-wise differentiation of iPSCs have been typically limited to the optimization of soluble factors. In this regard, we investigated how temporally varied substrate sti ...[more]

PMID: 28962663

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Project description:Recent studies suggested that embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) may represent different pluripotent states as defined by gene expression profiles and differentiation potential. Here we addressed a contribution of a lineage stage-specific donor cell memory in modulating the functional properties of iPSCs. iPSCs were generated from hepatic lineage cells at an early (hepatoblast-derived, HB-iPSCs) and end stage (adult hepatocyte, AH-iPSCs) of hepatocyte differentiation as well as from mouse fetal fibroblasts (MEF-iPSCs) using a lentiviral vector encoding four pluripotency-inducing factors Oct4, Sox2, Klf4, and c-Myc. All resulting iPS cell lines acquired iPSCs phenotype as judged by the accepted criteria including morphology, expression of pluripotency markers, silencing of transducing factors, capacity of multilineage differentiation in teratoma assay and normal diploid karyotype. However, hepatoblasts were more susceptible to reprogramming than either AH or MEF, and HB-iPSCs were more efficient in directed differentiation towards hepatocytic lineage as compared to AH-iPSCs, MEF-iPSCs or mESCs. Extensive comparative transcriptome analyses of the early passage iPSCs, donor cells and mESCs revealed that despite global similarities in gene expression patterns between generated iPSCs and mESCs, HB-iPSCs retained a transcriptional memory (7 up- and 20 down-regulated genes) typical of the original cells. Continuous passaging of HB-iPSCs abolished most of these differences including a superior capacity of hepatic re-differentiation. These results suggest that retention of lineage stage-specific donor memory in iPSCs may facilitate differentiation into donor cell type. The identified gene set may be helpful to improve hepatic differentiation for therapeutic application in liver disease modeling. A total of 200 ng RNA from four independent biological replicates of MACS-sorted mESC and iPSC were linearly amplified according to manufactures’ specification (Ambion, Austin, Tx,). For in vitro transcription (IVT), reactions were incubated for 16 h at 37ºC. The efficiency of the single round amplification was measured by NanoDrop (ND1000, Thermo Scientific). Hybridization, washing, detection (Cy3-streptavidin, Amersham Biosciences, GE Healthcare), and scanning were performed on an illumina iScan system (Illumina) using reagents and following protocols supplied by the manufacturer. The biotinylated cRNA (750 ng/sample) was hybridized on Sentrix beadchips human Ref-8v3 for 18 h at 58ºC while rocking (5 rpm).

Dataset Information

Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation.

Background

Methods

Results

Conclusions

Publications

Lineage- and developmental stage-specific mechanomodulation of induced pluripotent stem cell differentiation.

Similar Datasets

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