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Stable Long-Term Liver Stem Cell Culture Achieved

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Review of “Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver” from Cell by Stuart P. Atkinson

Successful in vitro culture and replication of functional human hepatocytes has confounded top researchers for many a year. Despite their high replicative capacity in vivo [1], in vitro¬ cultivation times are restricted to around one week, representing a 10-fold expansion of cells [2]. Investigations in mice are much further along, with existing strategies [3] allowing the long-term expansion of Lgr5-positive mouse liver stem cells [4] which can give rise to a plentiful source of adult liver cells for multiple different applications. Now, the laboratory of Hans Clevers (Hubrecht Institute-KNAW, Utrecht, Netherlands) has successfully adapted this mouse-based strategy to the human liver culture system, and they report their exciting findings in the January issue of Cell [5].

The researchers initially optimized their previously described mouse liver medium (ERFHNic [3]) to support human liver cultures for a prolonged period, without the loss of the all-important LGR5-positive stem cells. They found that enhanced Wnt signaling, alongside cAMP activation and TGF-β inhibition, inhibited liver differentiation and promoted the long-term expansion of stem-cell laden organoid cultures. Organoids are miniature 3D representations of an organ which can mimic the mature organ in many useful ways. The group also discovered, contrary to other studies [6, 7], that bile duct cells (EPCAM-positive), and not hepatocytes (EPCAM-negative), initiated organoid growth and therefore represented ductal and hepatocyte cell producing liver stem cell. Transcriptional analyses then found that organoid cells expressed stem cell markers (PROM1 and LGR5), ductal markers (SOX9, OC2) and hepatocyte markers (HNF4a), but not markers of mature hepatocytes (Albumin or CYP3A4). 

To differentiate organoid cells, the researchers added BMP7, which promotes hepatocyte proliferation in vivo [8], to the organoid cultures for 5-7 days prior to the inhibition of Wnt-signaling and cAMP activation, in addition to the Notch inhibitor DAPT, FGF19 and dexamethasone. This differentiation medium promoted organoid cells to take on a hepatocyte-like morphology, express hepatocyte markers at high levels, accumulate glycogen and load low density lipoprotein into the cell. Differentiated cells also excreted albumin and bile acid salts, exhibited similar CYP3A4 activity to freshly isolated hepatocytes, and demonstrated evidence of hepatocyte-like detoxifying capabilities. Overall, these organoid-derived cells displayed all the expected activities of a functional hepatocyte and functioned better than the reference hepatocyte cell line HepG2. Excitingly, these cells also demonstrated long-term liver engraftment in a mouse model of acute liver damage, with human hepatocyte-derived Albumin and α-1-antitrypsin protein found in serum within 7–14 days after transplant, at a level that remained stable for ~60 days in 5/6 mice and for ~120 days in 2 of the 5 grafted animals. 

The group then went on to study the effectiveness of patient-derived organoids to model human liver disease, specifically studying biopsies from patients with α1-antitrypsin (A1AT) deficiency and Alagille syndrome (AGS). Organoids derived from the A1AT-deficiency patient biopsy grew, differentiated and behaved normally for 4 months, producing cells broadly similar to healthy donor-derived organoid cultures. However, the differentiated cells did mimic the in vivo occurrences of A1AT deficiency, such as the formation of A1AT protein aggregates, endoplasmic reticulum stress and increased apoptosis. The organoid culture generated from an Alagille syndrome (AGS) patient biopsy, which results in structural defects of the biliary tree, also appropriately modelled disease development,

This encouraging study suggests that stem cell-derived liver organoids can effectively model normal liver function and liver-specific diseases with relative ease. This is encouraging news for liver therapeutics, with a host of new patient-specific therapeutic options now on the table, including liver regeneration, disease mechanism elucidation, cell replacement therapy, toxicology studies and drug testing. 

References

  1. Michalopoulos GK The liver is a peculiar organ when it comes to stem cells. Am J Pathol 2014;184:1263-1267.
  2. Shan J, Schwartz RE, Ross NT, et al. Identification of small molecules for human hepatocyte expansion and iPS differentiation. Nat Chem Biol 2013;9:514-520.
  3. Huch M, Dorrell C, Boj SF, et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 2013;494:247-250.
  4. Huch M, Bonfanti P, Boj SF, et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. The EMBO journal 2013;32:2708-2721.
  5. Huch M, Gehart H, van Boxtel R, et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 2015;160:299-312.
  6. Schaub JR, Malato Y, Gormond C, et al. Evidence against a stem cell origin of new hepatocytes in a common mouse model of chronic liver injury. Cell reports 2014;8:933-939.
  7. Yanger K, Knigin D, Zong Y, et al. Adult hepatocytes are generated by self-duplication rather than stem cell differentiation. Cell Stem Cell 2014;15:340-349.
  8. Sugimoto H, Yang C, LeBleu VS, et al. BMP-7 functions as a novel hormone to facilitate liver regeneration. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21:256-264.