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FEATURES

1. HepaRG is a highly stable cell lineHepaRG-karyotype-at-passage-12png
HepaRG karyotype at passage 12.

 

Progenitor cells  Progenitor cellsin

2. HepaRG is infinitely proliferating HepaRG proliferating progenitors HepaRG™ proliferating progenitors

HepaRG-chart-growth-at-passage-16


Asub-diploid karyotype showing only few alterations. This karyotype is highly stable for up to 20 passages.

An homogenous population of undifferentiated hepatic progenitors with morphological and functional characteristics of early hepatoblasts.

Note: The master and working banks set by BIOPREDIC allow to ensure a very high reproducibility of cell batches provided to customers.
An active proliferation rate with a doubling time of around 24h. At a seeding density of 2.7x104 cells/cm², new passages can be performed with high reproducibility every 2 weeks, up to 20 passages.

 Above chart displays the representative growth kinetic of HepaRG™ cells at passage 16; progenitors were seeded at 26000/sqcm in wells of 12 well-plates in William's E medium enriched with 10%FCS



 3. HepaRG cells can undergo a complete programme of hepatocyte differentiation

Complete differentiation programme
Complete differentiation programme

One week after seeding, confluent HepaRG cells start to commit into either hepatocyte (coloured in red in the picture above) or biliary differentiation pathways (coloured in grey).

Enriched HepaRG heptocytes population
Enriched HepaRG heptocytes population,

After 2 weeks, a mixed population of 2 types of cells, namely hepatocyte-like colonies surrounded by clear epithelial cells corresponding to primitive biliary cells, form a confluent monolayer which can be maintained in this stable form for several weeks in the presence of 1.7% DMSO.

Undifferentiated HepaRG cells
Fig.A: Undifferentiated HepaRG cells
F-actin is represented in red


Hepatocytes colonies
 are recognized thanks to the formation of numerous bile canaliculi (Fig B in red), characteristic of highly polarized, mature hepatocytes.

Differentiated HepaRG cells
Fig.B: Differentiated HepaRG cells
DNA labeling is represented in blue

However, because of the difficulty represented by selectively counting the hepatocyte competent cells and quantifying their peculiar activities with high efficiency, a specific protocol has been designed in order to increase hepatocyte selection in culture from confluent mixed HepaRG™ cell populations, thus allowing the scientists to reproduce adult human hepatocyte primary culture.

HepaRG™ cells express all main hepatic functions characterizing mature normal hepatocytes, including:

- detoxication function linked to hydrolyis and conjugation [Kanebratt KP 2008; Aninat C,2005]nitrate elimination [Hoechstra R, 2013, 2011]bile salt conjugation [Hoekstra R, 2013; Caron S. 2013]

- plasma protein production (albumin, transferrin, haptoglobin..) [Gripon P, 2002; Parent R., 2008; Klein S., 2013]

- energetic pathways linked to glucose (neoglycogenesis, glycolysis) and lipids (fatty acid) metabolism [Madec S 2011; Samanez CH., 2012; Plée-Gautier E. 2012]

- iron metabolism [Do Th., 2013].


Transferrin

Transferrin (in green)

CYP3A4 (green), F-actin (red)
CYP3A4 (green), F-actin (red)


Albumine
Albumin (in green and nuclei in blue)

Urea
Urea, 24hours


Asialoglycoprotein Receptor 1
Asialoglycoprotein Receptor 1

Glutamine
Glutamine, 24hours


Glycogen accumulation in HepaRG hepatocyte colonies
Glycogen accumulation in HepaRG hepatocyte colonies after 1 week confluence with 1.7% DMSO

 


 4. HepaRG expresses stem cell properties including transdifferentiation

HepaRG cells show a unique property of transdifferentiation through reversion to bipotent progenitor status [Cerec et al., 2007]Hepatocytes seeded at low density

  • It has been demonstrated that selectively collected hepatocyte-like, 98% positive CYP3A4 cells (represented in green in the figure below), seeded at low density (0.1 x104/cm2), display a great plasticity
  • correlated with a rapid decrease of liver specific functions related to CYP3A4 extinction in parallel to an increased expression of the CK19 progenitor marker (represented in red in the figure below).
  • After one set of divisions (day 15), cells reach confluence again and cords of granular hepatocytic cells re-emerge, surrounded by biliary-like cells (day 30).

Both hepatocyte-like and biliary-like cells can transdifferentiate through transient bipotent hepatic progenitor status.
Transdifferentiation is presumably associated to stemness of progenitor cells.



Pure HepaRG™ hepatocytes seeded at low density


 

 5.Bipotent progenitors transiently express stem cell properties:

HepaRG cells, P16. 0 TGF-b, 5 days
0 TGF-b, 5 days

Softness and plasticity
Plasticity is also preserved in differentiated cells.

HepaRG cells, P16: 2 weeks differentiated cells maintained for 5 days with 2.5 ng/ml TGF-b, loose their epithelio differentiated status and undergo transition to mesenchymal cells (EMT).

Cytoskeleton reorganization is observed as well as disappearance of biliary poles.

HepaRG cells, P16 - 2,5ng/ml TGF-b, 5 days
2,5ng/ml TGF-b, 5 days

Pluripotentiality
When HepaRG cells display bipotent proliferating progenitor phenotype, they share:
- with embryonic stem cells expression of OCT3/4,
- with oval cells expression of NCAM, ABCG2, CK18, and CK19,
- with hematopoietic stem cells expression of CD34, Thy1, Flt-3, c-Kit, IL-3R and LIF-R.

b-galactosidaseb-galactosidase

Self renewal / Senescence 

Progenitor cells are immortalized, i.e. they escape from aging process. Two pathways at least are known to be involved: hTERT expression and p27/p21 kinase inhibition. Noteworthy, p53 is free of mutation [Jossé R., 2012; Lereau M., 2012; Doktorova TY., 2013], while c-myc, p27 and p21 are normally regulated.

Nevertheless, when progenitors engage themselves into differentiation programme, giving rise to hepatocytes, these cells gradually accumulate aging markers such as an increased level of b-galactosidase and decreased hTERT activities as in normal hepatocyte primary cultures.
 
 


Protocol of cell line maintenance

Protocole cell line maintenance

Every 2 weeks, confluent HepaRG® progenitor monolayers are detached by 3-5min incubation with trypsin solution 0.05%. Cells are collected in William’S E medium containing insulin (4µg/ml) and hydrocortisone hemysuccinate (50µM) and added with 10%FCS.

After cell counting, they are seeded in the same medium at 20x103/cm2 and placed in a CO2 incubator at 37°. Medium is renewed every 2 or 3 days thereafter. Progenitors actively grow up to 5-6 days and are highly confluent after 2 weeks.

At this stage, they can be used either for a new trypsin treatment and a new passage in order to maintain the cell line, or incubated in the same medium added with 1.7%DMSO which favours the cells undergoing complete hepatocyte differentiation programme.

Within 3-4 days, two cell types are easily recognized: the one corresponding to hepatocyte colonies and the other one corresponding to primitive biliary cells.


 

Main applications

  • Sugar and lipid metabolism, physiopathology [Caron S. 2013]
  • Drug metabolism and toxicology [Guillouzo A., 2007; Lübberstedt., 2011; Andersson TB., 2012]
  • Viral infection [Gripon P., 2002, Glebe D., 2007; Hantz O., 2009; Sureau C 2010; Xie Y, 2010]

 

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