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iPSC-derived Hepatic Stellate Cells

DefiniGEN have generated the first commercially available iPSC-derived HSCs

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The liver is an organ of crucial importance, playing a central role in metabolism, detoxification, and immune function. While the primary cell type in the organ are parenchymal hepatocytes, other non-parenchymal cells are key to its functionality. 

Hepatic stellate cells (HSC) are pericyte-like cells that reside within the liver sinusoids, in an area known as the space of Disse. Recognized in healthy livers for their role in storing vitamin A, HSCs exhibit remarkable plasticity, transitioning between quiescent and activated states in response to liver injury.

Chronic liver injury can result in fibrosis, which subsequently leads to cirrhosis and hepatocellular carcinoma. Because this dysregulated HSC activation underlies the pathogenesis of hepatic fibrosis, HSCs are a potential area to target to identify novel therapeutics for alleviating liver fibrosis and restoring hepatic homeostasis.

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Limitations in Animal, Primary Cell and Immortalized Cell Models

Primary human hepatic stellate cells

Primary human hepatic stellate cells (pHSCs) are considered the gold standard in vitro model for fibrosis-related studies. However, these cells are difficult to isolate, demonstrate heterogeneity among liver donors, and the considerable differences between the diverse isolation processes increases batch-to-batch variation. In addition, pHSCs rapidly lose their quiescent features, they have a limited life span, and yield contamination with inflamed liver macrophages is often observed. 

 

Hepatic stellate cell lines

To overcome some of the limitations of pHSCs, several immortalized rodent and human hepatic stellate cell lines (e.g., HSC-T6, LX-2) have been developed. These cell lines express smooth muscle actin filaments (α-SMA), esterify retinol, and respond to mediator of liver fibrogenesis (e.g., TGF-1), thus offering good HSC model alternatives. However, these cell lines show an activated HSC phenotype in culture, lack key HSC functional characteristics, and are prone to genotypic and phenotypic drifts following pro-longed culture and passage. In addition, rodent-derived HSC lines demonstrate species-specific differences, downstream compromising data translatability. 

Key Features and Benefits of Our Hepatic Stellates

The development of induced pluripotent stem cells (iPSCs) has created a new opportunity in in vitro cell modelling, offering a promising alternative for the generation of fully functional liver cell types with unlimited expansion capacity, phenotypic stability and healthy karyotype.

DefiniGEN have generated the first commercially available iPSC-derived HSCs, which demonstrate the following advantages:

High purity

Our iPSC-derived hepatic stellates are not sourced from tissue and therefore aren’t mixed with other cell types

Single donor

Sourced from a single donor: Ensuring there is no batch-to-batch variation 

Quiescent status

Which can be activated upon treatment with liver fibrogenesis mediators (TGF-1)

Cell markers

Expression of key hepatic stellate cell markers: e.g., Desmin, GFAP, PDGFRs, α-SMA and Collagen I

Collagen I secretion

 

Healthy karyotype

Technical data

Figure 1: Key HSC markers

DefiniGEN iPSC-derived Hepatic Stellate Cells (Opti-HSC) express key HSC markers at gene and protein level

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Figure 1A: mRNA levels of HSC markers (Desmin, PDGFRb, ALCAM, PCDH7, ACTA2, and COL1A1) in DefiniGEN Opti-HSC following 2 weeks of iPSC differentiation and primary HSC (pHSC).

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Figure 1B: Protein levels of the key HSC markers Desmin and GFAP in DefiniGEN Opti-HSC.

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Figure 1C: Low protein levels of the HSC-activation markers Collagen I and αSMA in DefiniGEN Opti-HSC, indicative of their quiescent status.
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Figure 1D: mRNA levels of hepatocyte markers (ALB, A1AT, HNF4A) in iPSC-derived Opti-HSC and Opti-HEP as well as pHSC and primary human hepatocytes (PHH). Data are presented as mean±SEM of n=2 independent experiments. mRNA expression data were normalized to 18S rRNA.

Figure 2: Quiescent Opti-HSC

Quiescent Opti-HSC are successfully activated using pharmacological and mechanical stimuli

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Figure 2A: Simplified timeline of the differentiation and activation of iPSC-derived Opti-HSC using pharmacological and mechanical stimuli (TGFβ treatment or plastic attachment).

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Figure 2B: mRNA levels of the HSC-activation marker COL1A1 in vehicle-treated, TGFβ-treated, or re-plated DefiniGEN Opti-HSC for 5 days. 

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Figure 2C: Protein levels of Collagen I in vehicle-treated, TGFβ-treated, or re-plated DefiniGEN Opti-HSC for 5 days. Data are presented as mean±SD of n=3 technical replicates. mRNA expression data were normalized to 18S rRNA. Cells were counterstained with DAPI.

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Figure 2D: mRNA levels of the HSC-activation marker ACTA2 in vehicle-treated, TGFβ-treated, or re-plated DefiniGEN Opti-HSC for 5 days.
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Figure 2E: Protein levels of αSMA in vehicle-treated, TGFβ-treated, or re-plated DefiniGEN Opti-HSC for 5 days. Data are presented as mean±SD of n=3 technical replicates. mRNA expression data were normalized to 18S rRNA. Cells were counterstained with DAPI.

Figure 3: Maintain cell signature 

iPSC-derived hepatic stellate cells maintain their cell signature post-cryopreservation

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Figure 3A: 10x objective. >85% cell viability. Standard hepatic stellate morphology. >85% double positive cells for GFAP and αSMA.

DefiniGEN cryopreserved iPSC-derived hepatic stellate cell morphology 24 hours post thaw.

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Figure 3B: Protein levels of the key HSC markers GFAP, αSMA, and Collagen 1 in cryopreserved DefiniGEN Opti-HSC.

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