Peer review of: Mechano-modulatory synthetic niches for liver organoid derivation (2019)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
One of corresponding authors, Dr. Lutolf, previously reported chemically defined matrices for intestinal 
stem cell culture and organoid derivation (ref 7, Nature 2016). In this study (NCOMMS-19-29398),  the 
authors did the similar experiments using chemically defined hydrogels (inert poly(ethylene glycol), 
PEG) and hepatic cells. The authors insist that they succeeded to prepare mouse and human liver 
organoids through actomyosin-independent YAP activation. The aim of this study is clear, however, 
their results are not enough to conclude liver organoid derivation and its molecular mechanism. 
Therefore, I can not recommend the present manuscript for publication. 
 
Major comments 
1. The liver is composed of several types of cells including hepatocyte, stellate, biliary, and Kupffer 
cells etc. So, the authors’ definition of liver organoid is not enough. The authors should show cell-
types of the organoid by FACS analysis (Ouchi et al. Cell Metabolism 2019). 
 
2. In Fig. 3, the authors used only one inhibitor, blebbistain, as acto-myosin contractility inhibition. 
The authors should use another inhibitor, ROCK inhibitor Y-27632. 
 
3. Dasatinib inhibits Src family member kinases, which affect actin cytoskelton. The authors should 
explain the pathway(s) of the actomyosin-independent YAP activation in detail. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
Sorrentino et al. present results on a synthetic poly(ethylene glycol) (PEG) hydrogel for the derivation 
of both mouse and human hepatic organoids. This elegant study demonstrates equivalent hepatic 
organoid generation between the synthetic hydrogel and Matrigel. Furthermore, this synthetic system 
overcomes major limitations of tumor-derived Matrigel and represents a significant technical advance. 
The authors also show that the stiffness of PEG hydrogel modulates hepatic organoid development and 
provide results implicating Src-family kinases and YAP in this response. Results for fibrosis-associated 
genes in stiffer hydrogels are presented, but this data is rather preliminary and not particularly 
compelling. 
 
1. Are the results unique for PEG hydrogels? Do hepatic organoids form in alginate (with/without  RGD 
peptides) or collagen gels? 
 
2. Similar levels of organoid formation and differentiation are observed among hydrogels presenting 
laminin-1, fibronectin, and RGD peptide (Fig. 1b, d). Are the densities of these adhesive motifs 
equivalent across the groups? Do the organoids deposit an extracellular matrix at the hydrogel 
interface? 
 
3. Immunostaining for hepatic markers and differentiation should be assessed for the murine 
organoids established without Matrigel culture (Fig. 1f, S1). 
 
4. Fig. 3a shows differences in organoid formation efficiency as a function of hydrogel stiffness. The 
authors should include results for proliferation and immunostaining analyses for hepatic differentiation 
markers to more fully characterize these observations. Furthermore, the stiffness of Matrigel should 
be reported and the results discussed in this context. 
 
5. Results for fibrosis-associated genes in stiffer hydrogels are interesting (Fig. 4), but this data is 
rather preliminary and not particularly compelling. Results at protein level and matrix deposition 
should be included to support the gene expression data. 
6. The statement “abnormal ECM stiffness is sufficient to decrease the liver progenitor proliferative 
capacity” (line 161) should be supported with data. 
 
7. For the results with freshly isolated liver biopsies from patients (without  Matrigel, Fig. 5f), 
immunostained images for hepatic markers should be presented and compared to those grown in 
Matrigel. 
 
 
 
Reviewer #3 (Remarks to the Author): 
 
In this manuscript the authors report chemically defined hydrogels (PEG-RGD) for the efficient 
derivation of both mouse and human hepatic organoids. They show these organoids can be readily 
differentiated into hepatocyte-like cells. Moreover, they identify Src family of kinases (SFKs) rather 
than actomyosin contractility, mediated yes-associated protein 1 (YAP) activity that is required for 
hepatic organoid growth. Finally, they generate clinically relevant human hepatic organoids from 
human patients with PEG-RGD. The manuscript is clear and well-written. However, there are 
important issues that should be addressed to improve the manuscript: 
 
1. Although PEG-RGD enable to support the growth of hepatic organoids, the long-term expansion 
potential of these organoids is not clear. The authors only show the relevant data of passage 3 (figure 
1f). There is no additional experiments, which convincingly shows that the PEG-RGD-derived 
organoids can be expanded for an extended period of time. After all, hepatic organoids in MATRIGEL 
could expand for more than 6 months with a consistent doubling time in a previous study (PMID: 
25533785). 
2. The authors should deeper analyze the identity of organoids. Comparing the whole genome 
transcriptome of PEG-RGD-derived organoids with the MATRIGEL-derived organoids would be needed. 
3. Regarding the analysis of hepatocyte differentiation capacity of PEG-RGD-derived organoids, the 
authors should include normal human/mouse hepatocytes as positive controls (adult and fetal if 
possible) in their gene expression assays and in functional assays (figure 2a, 2e, 2f, figure s2b and 
s4b). Without these controls, the data presented has limited significance. 
4. After differentiation, the PEG-RGD-derived organods seemingly show less ALB expression than 
MATRIGEL-derived organoids, evidenced by immunostaining (figure 2b). Does this mean that the PEG-
RGD-derived oragnoids have compromised hepatocyte differentiation potential? Moreover, the 
organoids in expansion medium already express Ecad and HNF4A before differentiation. Therefore, 
these markers are not appropriate for assessing the extent of hepatocyte differentiation. To clarify this 
situation, quantitative data along with the immunostainings should be provided in figure 2b-2d. 
Additionally, the analysis of hepatocyte differentiation of MATRIGEL-derived organoids should be as a 
control in figure 2 and figure s2. 
5. The authors demonstrate that organoid formation and growth in PEG-RGD depend on YAP 
activation. Whether the YAP activators such as lysophosphatidic acid (LPA) and sphingosine-1-
phosphate (S1P) could improve the organoid formation efficiency in PEG-RGD of various stiffness? 
6. Increased stiffness (4kb) result in decreased organoid proliferation without affecting YAP activation, 
suggesting the existence of other pathways controlling liver organoid growth in response to increased 
stiffness. Recently, Aloia L et al  demonstrated that both of YAP–Hippo and ErbB–MAPK signaling 
pathways play crucial role in adult cholangiocyte-derived organoid formation (PMID: 31685987). Are 
ErbB-MAPK signaling involved in regulation of organoid formation in aberrant stiffness (4kb)? This 
needs to be further addressed. 
7. Finally, the authors show that clinically relevant human stem/progenitor cells can be grown in vitro 
without any requirement of animal-derived matrices and state that this may open exciting 
perspectives for liver organoid-based clinical applications. Because large number of cells are need for 
clinical applications, it would be good to show the expansion potential of these biopsy-derived human 
liver organoids. Besides, whether these oragnods could be frozen and thawed is also critical. 
Karyotype analysis of biopsy-derived human oragnoids in PEG-RGD of early and later passages should 
be provided. 
8. In figure 5c and 5d, please exchange the label PEG-RGD (HDM) with PEG-RGD (HEM).