Peer review of: Characterization of a pluripotent stem cell-derived matrix with powerful osteoregenerative (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The manuscript by McNeill et al. derives an osteogenic cell-matrix and tests its bone anabolic 
activity in a calvarial defect model in mice. The premise of the study is well-justified as few 
therapeutics exits that exhibit anabolic activity in bone. The study design is also rigorous, and the 
presented data is of high quality. The statistical analyses appear appropriate and methods are 
sufficient to allow the work to be reproduced. However, several concerns were identified related to 
specific methods and data sets that should be addressed prior to publication. 
 
Major concerns 
1. The authors present a rigorous set of data characterizing the tri-lineage potential of ihMSCs and 
BM-MSCs. However, the desire to characterize their immuno-suppressive and anti-inflammatory 
activities is questionable given the premise of the study, and the data presented in this regard are 
modest at best. Therefore, it would seem prudent to relegate these data (Figure 1e, f) to a 
supplementary figure. One concern is that CFSE-based labeling shows a complete inhibition of cell 
proliferation by ihMSCs and a loss of label in control cells, which is atypical. The authors should 
reduce the incubation time/CFSE labeling to fall within the dynamic range of the assay and include 
different ratios of effector to target cells. BM-MSCs and an unmixed PBMC control to determine the 
degree of stimulation should also be included. 
 
2. The authors demonstrate that OEihMSCs generate higher yields of ECM as compared to 
OEhMSCs, which is enhanced by GW9662, without altering protein content. Assuming the 
increased mass represents GAGs, it would be useful to demonstrate this by quantitative analysis. 
 
3. Data in Figure 3a,b is curious in that OEihMSC and OEhMSC1 cluster together but GW9662 
treatment increases the distance between these samples. This likely reflects the fact that GW9662 
treatment reduces to zero the number of ECM proteins shared by these two populations (Fig. 3a). 
GW9662 treatment increases the number from 1 to 3 of proteins unique to OEihMSCs. What are 
these proteins and were they tested for their osteogenic promoting activity? 
 
4. The authors incubate that “treated ihOCM upregulated the activity of ALP and the early 
osteogenic biomarker osteoprotegerin (OPG) by attached ihMSCs” but the legend to Figure 3g 
states that “hMSCs were attached to ihOCM generated in the presence of GW9662 (grey) or 
vehicle (white)”. Please clarify if ihMSCs or hMSCs were used. In either case, it would seem 
appropriate to test both matrices and populations in combination to validate the superior activity 
of ihOCM in this assay. Does the matrix induce any activity in the absence of osteogenic 
stimulants? 
 
5. Data based on knockdown studies appear rigorous, but it may be useful to evaluate effects on 
apoptosis/viability, e.g. do cells grow more slowly or is their rate of cell death higher? This may be 
important in evaluating the mineralization data form Figure 4g, e.g. why does mineralization fall 
off between D14 and D21 in the KD12 sample. 
 
6. All studies evaluate early markers of mineralization, such as ALP. Is it valid to look at markers 
of secondary mineralization (late stage), such as osteocalcin, Mepe, etc.? 
 
7. Results of calvarial defect model studies are compelling. Did the authors evaluate control mice 
with defects for longer time periods to ensure the 4mm hole represents a critical size defect, e.g. 
no significant healing? Effects of the ihmatrix (assume this is equivalent to ihOCM) is remarkable 
and inclusion of an additional timepoint may be beneficial to demonstrate complete healing vs. 
other formulation tested. 
8. How was healing index calculated? Also, since authors are proposing an osteogenic matrix to 
accelerate fracture healing, they should provide a rationale for using the calvarial defect model 

(non-weight bearing bone) vs. a tibia burr hole defect model (weight bearing). Ultimately, the 
matrix should be tested in a surgical defect model to evaluate the integrity/strength of the 
repaired bone. 
 
Minor comments: 
 
1. In Suppl Figure 2b, graphs illustrating results of qPCR analyses employ different scales, which 
makes evaluation of differences between groups difficult. It is suggested the authors use a single 
scale, which would magnify differences in gene induction among the different cells tested. 
 
2. Data in Suppl Figure 3a demonstrating effects of troglitazone or CCT032374 alone or in 
combination on ihMSC adipogenic is qualitative and therefore subjective. Quantative analysis 
should be performed to asses stimulatory effect of each agent and if they work additively in 
combination. 
 
3. In Figure 2b, PPARgamma2 protein abundance in normalized to GAPDH, but the control used in 
the blot is beta-actin. Same error appears in Figure 2d with beta-catenin. Please clarify. 
 
4. Is ihmatrix equivalent to ihOCM? Please keep terminology consistent as it is confusing enough. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This manuscript reports on a study in which hMSC were derived from hiPSC, and were then used 
to generate an extracellular matrix (ihOCM). This matrix and the cells that produced it were 
carefully characterized and were compared to bone marrow-derived MSC and the matrix that they 
produce. The role of Wnt signaling and PPARg in the osteogenic capacity of the ihMSC was 
investigated, as was the role of Col VI and Col XII proteins, which were found to be more 
abundant in ihOCM than in MSC-produced matrix. The effect of ihOCM on bone regeneration in a 
calvarial defect in the nude mouse was then assessed, relative to controls, and with/without the 
addition of cells. The main findings of the paper relate to the composition and osteogenic 
properties of the ihOCM, with particular emphasis on the roles of collagen VI and XII. The authors 
assert that “Based on these findings, we propose that ihOCM may represent an effective 
replacement for the current gold standards, autograft and BMP products, used commonly in bone 
tissue engineering.” 
 
This paper is of interest to those in the regenerative medicine community who study the biology 
and application of progenitor cells, in particular those interested in the application cell and 
biomaterials to bone healing. The manuscript is generally well prepared, the data are for the most 
part clearly presented, and the study is technically sound. The work on characterizing the ihMSC 
and ihOCM is particularly comprehensive. However there are several issues with this paper in 
relation to its place in the field: 
 
1] The use of cell-derived extracellular matrix to guide cell function and to ultimately potentiate 
regeneration has been studied widely. More specifically, the use of MSC-derived matrix to promote 
osteogenesis has been previously reported by several groups, including in previous publications by 
the authors (Refs 20-22). The main innovation in this paper seems to be the use of iPSC-derived 
MSC, the derivation of which is also widely reported in the literature, for similar purposes. The 
impact of this paper on the field of bone regeneration is therefore not evident, and it would be 
helpful to explain how this study significantly extends the preceding work by a number of groups 
internationally. 
 
2] One aspect of this study’s approach that is highlighted is that the use if iPSC as “a theoretically 
limitless and reproducible source of cells” (p. 3). However, it is now established that iPSC from 

different donors can exhibit practically significant variability (see e.g. Nature 2017, 546:370-375) 
and furthermore that derivation of iPSC is not always consistent. For a proposed patient-specific 
therapeutic approach, this would seem to be problematic. The reported study uses “a single line of 
iPSCs” (p. 11), which calls into question the broader applicability of the approach, in particular 
since the authors clearly point out the inter-donor variability of MSC. The study would be improved 
by validating that iPSC-derived MSC are in fact more consistent across donors, as this seems to be 
a key to this approach being an improvement. 
 
3] The data in the paper focus largely on the properties of the ihMSC and the composition of the 
resulting ihOCM, including the molecules and mechanisms that are involved in osteogenesis, which 
appear to be the same as in bone marrow-derived MSC. These in vitro studies are quite 
comprehensive and well done, and provide good evidence for the conclusions the authors draw 
regarding the in vitro characterization of the cells and matrix. However, the title of the paper 
highlights the “osteoregenerative capabilities” of the ihOCM, and relatively few data on bone 
regeneration are reported; of the 43 panels of data presented, only 7 contain data on bone 
regeneration, and these are not very comprehensive. The paper’s main point appears to be that 
iPSC-derived MSC are similar to other MSC in terms of the components and mechanisms by which 
they promote osteogenesis, and may be more potent (if it can be shown that other/all ihMSC 
behave similarly). This is an interesting finding, though not one that is reflected in the title of the 
paper. 
 
4] The manuscript is generally very well-written and clear, and the Methods are adequately 
described; the exception is the derivation of the iPSC, which is important but included only by 
reference to a previous paper (which is incorrectly cited; it is presumably Ref 17). The Discussion 
section is quite repetitive of the Results section, and could be made more concise by focusing on 
the impact of the findings and their relevance to the current state of the field. The final paragraph 
of the Discussion is not compelling in terms of describing how these findings will influence the 
thinking of others in the field. There are a few awkward phrasings and terminology; e.g. use of the 
term “holy grail” is rather colloquial for a scientific manuscript, the authors refer to “stress-
processes” (perhaps they mean “stress fibers”?), there is a typo in the legend to Figure 2.