Mesenchymal Stromal Cells (MSCs) Isolated from Various Tissues of the Human Arthritic Knee Joint Possess Similar Multipotent Differentiation Potential

Round 1

Reviewer 1 Report

The study explores MSC-like cells, and aims to deliver them to targeted sites for regenerative purposes. As such, it is very interesting, since it paves the way towards individualised treatment schemes. The confusing part is that there were five subjects, whose apparent tissues were harvested across various sites? Mixing in a sixth knee only adds to the confusion, as there is no system where – the sixth knee makes it impossible to properly assess this (due to the chance of varying amounts of arthropathy in two knees of the same person, which could have theoretically have different epigenetics reaction/exšpression, making these two sources possibly incomparable). Please try to limit this, or re-run, or if all that fails, list as a possible limitation. It would have been great if you could add a more junior person in the analysis, or at least show differences, or at least discuss this (assuming their better bio/regenerative potential). Although the expression of the protein is known to vary wildly, I liked the semi-quantitative approach, given it gets appropriately analysed. There is a broken line in the Statistical analysis section. The overall analysis may be a bit tricky here, due to the extremly small sample size. I dislike the use of KW test, I would have maybe suggested a more robust categorisation and the use of the chi-square test. The results are well within expectations, with the BM being the most active, and the other tissues being comparable. Slightly puzzling is the IPF, which I would have assumed to be maybe a bit more active than shown in Figure 1; this is later shown in line with the expectations in Figure 4, so it is slightly odd to see this discrepancy. Please refer to Primorac et al. study that utilises the adipose cells to do exactly this, in real patients, and compare with your results. Do discuss the advantages and limitations of selected tissues for harvesting, and establish the potential clinical translation of your findings. Do mention possible risks of the MCS, especially in heavy-duty tissues, like the knee joint. You mention KW test being used, but there are no P values given in the text? Please provde the exact P values, so we can decide about the meaning of these results. In general, the numerical part of the analysis is somehow downplayed, making this entire manuscript somewhat less clear. I think that overall, the authors did a good job, and that with a few of these minor changes the manuscrip can be accepted.

Author Response

Dear Reviewer,

please find our answers to your comments attached.

We highly appreciate your comments to our current manuscript and thank you for greatly improving it.

Regards

Dr. Mike Wagenbrenner

Author Response File: Author Response.pdf

Reviewer 2 Report

The article by Wagenbrenner et al. entitled "Mesenchymal stromal cells (MSCs) isolated from various tissues of the human arthritic knee joint possess similar 3 multipotent differentiation potential", shows a study comparing the differentiation capacity of MSCs from different tissues obtained from human arthritic knee joint, among them and with respect to MSCs obtained from bone marrow (BM-MSC). The study is interesting because it shows the possibility of obtaining MSCs from different tissues for therapeutic purposes through less invasive methods.  However, the work has important limitations, some of which are acknowledged by the authors. In my opinion, these limitations do not allow to obtain sufficient data to justify the title and conclusions of the study. The results do not show conclusive data that cells obtained from different tissues have similar properties to BM-MSCs.

Remarks:

1. Flow cytometry. The authors should have also studied the expression of hematopoietic markers (e.g. CD34, CD45 and CD14). MSCs should be negative for these markers.
2. Flow cytometry. Only results from one patient are shown. As well as the differentiation capacity of the cells obtained from all patients has been studied, the expression of MSC and hematopoietic markers should have been studied in all patients. The results shown indicate that at the level of CD73 expression, MSCs isolated from various tissues of the human arthritic knee do not fulfill the ISCT criteria. This is in contrast to what has been described in other studies. In fact, the authors have published a paper on MSC isolation from the anterior cruciate ligament, in which the cells obtained are CD73 positive like those derived from bone marrow (DOI: 10.1089/ten.tea.2010.0413). Moreover, as it has only been studied in one patient, it is not possible to know whether this result depends either of the selected patient or of the population of cells in those tissues.
3. Material and Methods: primers used. According to the analysis of the primers used by "primer blast", the amplicon sizes of several of them do not match those shown in Table 3. In addition, are the primers for COL2A1 correct?. The primers shown for COL1A1 amplify COL1A2, but not COL1A1.
4. The results show that the cumulative population doublings in BM-MSCs are higher than in other MSCs. Because of the large numbers of cells that are usually needed for cell therapy applications, this is a very important limitation for clinical utilization of MSCs isolated from the rest of the tissues. In addition, the work lacks a study on the passage number and expansion capacity that MSCs obtained from different tissues are able to support in culture. This is fundamental in the comparison of the therapeutic potential of these cells.
5. The authors claim in the discussion that MSCs obtained from different osteoarthritic knee joint tissues have the same differentiation capacity as BM-MSCs. To make this affirmation, a quantitative analysis of histological and immunohistochemical staining performed on cells differentiated to osteoblasts, adipocytes and chondrocytes should be performed.ç
6. Figure legends: do not repeat at the end of each figure the meaning of the abbreviations used. In the description of the figure, they are already described.

Author Response

Dear Reviewer,

please find our answers to your comments attached.

We highly appreciate your comments to our current manuscript and thank you for greatly improving it.

Regards

Dr. Mike Wagenbrenner

Author Response File: Author Response.pdf