Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes
- National Cancer Center Research Institute, Japan
- Keio University, Japan
- Nihon Pharmaceutical University, Japan
- Kobe Pharmaceutical University, Japan
- Tokyo Medical University, Japan
Figures
Figure 1
Morphological changes of hepatocytes in response to small molecule stimuli with/without FBS.
(A) Phase contrast images of APHHs cultured in the presence of YAC, which are used to obtain rat and mouse CLiPs. Insets indicate representative magnified images. (B) Phase contrast images of rat …
Figure 2 with 3 supplements
AC together with FBS support the expansion of IPHHs.
(A) WST assay assessing the effects of various combinations of Y, A, and C together with 10% FBS on proliferation of 8-month-old IPHHs (lot FCL). Absorbance at 450 nm was determined at D14 and …
Figure 2—figure supplement 1
Proliferative colonies observed in infant and juvenile PHHs.
Phase contrast images of PHHs obtained from infant donors (four lots) and a juvenile donor (one lot). Regions with spontaneous hepatic differentiation are magnified.
Figure 2—figure supplement 2
Characterization of FAC-cultured proliferative human hepatic cells.
(A) Flow cytometry of LPC surface markers for hCLiPs derived from lots DUX and JFC PHHs. (B) Flow cytometry of LPC surface markers for cryopreserved PHHs. The cells were thawed and stained with the …
Figure 2—figure supplement 3
Cell-cycle-related gene expression analysis by GSEA.
(A) GSEA demonstrating enrichment of cell-cycle-related gene sets in cells cultured in FAC in comparison with cells cultured in the presence of FBS at D14. (B) GSEA demonstrating enrichment of …
Figure 3 with 4 supplements
Comparative analysis of responsiveness to FAC of IPHHs and APHHs.
(A) Hierarchical clustering for whole transcriptome of IPHHs (red) and APHHs (blue). Two lots of APHHs (HC5-25 and 187271) and three lots of IPHHs were compared. (B) Gene expression of LPC marker …
Figure 3—figure supplement 1
GSEA comparing IPHHs and APHHs cultured in FAC for 7 days (KEGG database).
Pathways enriched (nominal p-value<0.05) in IPHHs compared to APHHs are shown.
Figure 3—figure supplement 2
GSEA comparing IPHHs and APHHs cultured in FAC for 7 days (Hallmark database).
Pathways enriched (nominal p-value<0.05) in IPHHs compared to APHHs are shown.
Figure 3—figure supplement 3
Morphological changes of APHHs and IPHHs which were cultured in FAC medium.
Note that HC5-25 APHHs formed progenitor-like colonies partly, while 187271 APHHs did not. Proliferating cells in 187271 culture were only non-hepatic cells as determined by microscopic observation. …
Figure 3—figure supplement 4
Individual plots of GSEA results for TGFβ, Wnt and mTORC1 signaling pathways.
Figure 4 with 1 supplement
FAC-cultured proliferative cells differentiate into mature hepatocytes in vitro.
(A) Phase contrast images showing the morphological changes of FAC-cultured human proliferative cells (lot FCL) treated with (Hep-i(+)) or without (Hep-i(-)) hepatic maturation-inducing factors. …
Figure 4—figure supplement 1
Characterization of proliferative human hepatic cells following hepatic maturation.
(A) Phase contrast images showing the morphological changes of hCLiPs derived from lots DUX and JFC upon hepatic maturation. (B) Quantified expression of BEC/LPC marker genes in hCLiPs derived from …
Figure 5 with 1 supplement
hCLiP-derived hepatocytes exhibit CYP enzymatic activity.
(A) Heatmap showing expression of CYP genes that were differentially expressed between Hep-i(-) and Hep-i(+) cells (≥1.5 fold change), as assessed by microarray analysis. Fold change was calculated …
Figure 5—figure supplement 1
Inducibility of CYP1A2 and CYP3A4 in Hep-i(+) cells.
(A) qRT-PCR analysis of the inducibility of CYP1A2, CYP2B6, and CYP3A mRNA expression. Gene expression levels were normalized against that of ACTB. Data are shown as one representative experiment. (B…
Figure 6 with 1 supplement
hCLiPs stably expand in vitro and retain their hepatic differentiation ability.
(A) Growth curves of hCLiPs from P0–10 (lots FCL and DUX) or P0–four or P0–5 (lot JFC). Each curve represents data obtained in independent experiments. Data in each plot indicate the cumulative cell …
Figure 6—figure supplement 1
Characterization of hCLiPs upon long-term culture.
(A) Phase contrast images of hCLiPs upon serial passage. Arrows indicate cells with a fibroblast-like morphology. (B) Surface marker profiling of hCLiPs upon serial passage. Data are from three …
Figure 7 with 2 supplements
FACS is useful for enrichment of hCLiPs, but does not prevent phenotypic deterioration of the descendant cells in the subsequent culture.
(A) Schematic representation of the experimental design for FACS of LPC marker+THY1- cells (hCLiP-enriched cells) and LPC marker-THY1+ cells (putative NPCs), and the subsequent evaluation by …
Figure 7—figure supplement 1
Detailed data for FACS of FCL-hCLiPs using antibodies for LPC markers and THY1.
Unsorted cells which were cultured for the same period with sorted cells are shown on the top.
Figure 7—figure supplement 2
Comparison of gene expression levels of FCL-hCLiPs just after sorting and their descendants which underwent another 2 week culture.
Figure 8 with 1 supplement
hCLiPs repopulate chronically injured mouse livers and contribute to reconstruction of the normal liver architecture.
(A) hALB levels in blood of cDNA-uPA/SCID mice. Each line indicates the level in an individual mouse. Colors denote the passage number of transplanted hCLiPs. (B) Representative images of …
Figure 8—figure supplement 1
Repopulation assay of FCL-hCLiPs at later passages.
(A) Blood hALB levels in cDNA-uPA/SCID mice which were transplanted with FCL-P4-hCLiPs (purple lines). Each line indicates the level in an individual mouse. cDNA-uPA/SCID mice transplanted with …
Figure 9 with 1 supplement
Human cells isolated from chimeric livers of mice transplanted with hCLiPs have mature functions.
(A) Phase contrast images of human cells isolated from chimeric livers of mice transplanted with hCLiPs. (B) Hierarchical clustering based on Euclidean distance of the entire transcriptome (27,459 …
Figure 9—figure supplement 1
Characterization of human cells isolated from chimeric livers of mice transplanted with hCLiPs.
(A) Heatmap showing expression of BEC/LPC marker genes, as assessed by microarray analysis. Each element represents normalized (log2) expression, as indicated by the color scale. Hierarchical …
Author response image 1
Author response image 2
Comparison between ProliHHs and hCLiPs in terms of in vitro growth rates.
(A) According to Figure S5A in Zhang et al., 2018, Early ProliHH and Late ProliHH underwent approximately 200 and 1000 fold expansion from the starting cell number. Note that our passage counting …
Author response image 3
Comparison between ProliHHs and hCLiPs in terms of their repopulation efficiency.
(A)Repopulation experiments in FRG mice using ProliHHs at early (P4) and late (P6) passages. Data are adapted from Supplementary Figure S6G (Zhang et al., 2018).(B)hCLiP (lot FCL) repopulation …
Tables
Table 1
Donor information of primary human hepatocytes (PHHs) used in this study.
| Cell type | IPHH | IPHH | IPHH | IPHH | IPHH | APHH | APHH | APHH | APHH | APHH |
|---|---|---|---|---|---|---|---|---|---|---|
| Lot | FCL | DUX | JFC | MRW | 187273 | HC7-4 | HC5-25 | HC1-14 | HC3-14 | 187271 |
| Age | 10 mo | 8 mo | 1 yr | 11 mo | 2 yr | 7 yr | 56 yr | 55 yr | 45 yr | 26 yr |
| Sex | Female | Male | Male | Male | Male | Male | Male | Male | Male | Male |
| Race | Hispanic | Caucasian | Caucasian | Caucasian | Caucasian | Caucasian | Caucasian | Caucasian | Caucasian | Caucasian |
| Cause of death | Anoxia/drowning | Anoxia/cardiovascular | Anoxia/second to blunt injury | Asphyxiation | N/A | Anoxia | Cerebrovascular Accident | Anoxia | Cerebrovascular Accident | N/A |
| CMV | - | - | - | + | N/A | + | + | - | - | N/A |
| HIV | - | - | - | - | - | - | - | - | - | - |
| HBV | - | + | + | - | - | - | - | - | - | - |
| HCV | - | - | - | - | - | - | - | - | - | - |
| EBV | - | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A |
| RPR | - | N/A | - | - | N/A | N/A | N/A | N/A | N/A | N/A |
| HTLV | N/A | N/A | N/A | - | N/A | N/A | N/A | - | - | N/A |
-
IPH: Infant primary human hepatocyte; APH: adult primary human hepatocyte; CMV: cytomegarovirus; HIV: human immunodeficiency virus; HBV: hepatitis B virus; HCV: hepatitis C virus; EBV: Epstein-Barr virus; RPR: rapid plasma reagin; HTLV: human T-cell leukemia virus; N/A: information not available.
Additional files
-
Supplementary file 1
All the gene sets enriched in FAC cells compared with FBS cells at D14 of culture (assessed by GSEA).
- https://cdn.elifesciences.org/articles/47313/elife-47313-supp1-v3.xlsx
-
Supplementary file 2
All the gene sets enriched in FAC cells at D14 of culture compared with D1 hepatocytes (assessed by GSEA).
- https://cdn.elifesciences.org/articles/47313/elife-47313-supp2-v3.xlsx
-
Supplementary file 3
All the gene sets enriched in FBS cells at D14 of culture compared with D1 hepatocytes (assessed by GSEA).
- https://cdn.elifesciences.org/articles/47313/elife-47313-supp3-v3.xlsx
-
Supplementary file 4
Significantly enriched gene sets (Nom p<0.05) in Hep-i(+) cells compared with Hep-i(-) cells (assessed by GSEA).
- https://cdn.elifesciences.org/articles/47313/elife-47313-supp4-v3.xlsx
-
Supplementary file 5
Significantly enriched gene sets (Nom p<0.05) in Hep-i(-) cells compared with Hep-i(+) cells (assessed by GSEA).
- https://cdn.elifesciences.org/articles/47313/elife-47313-supp5-v3.xlsx
-
Supplementary file 6
Significantly enriched (NOM p<0.05) gene sets in hCLiP-chimera-derived hepatocytes in comparison with PHHs.
- https://cdn.elifesciences.org/articles/47313/elife-47313-supp6-v3.xlsx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/47313/elife-47313-transrepform-v3.docx
