This proposal is focused on the human placenta as a high capacity source of stem cells that can be harvested both for therapeutic applications and for drug discovery approaches. In the first aim, the applicant proposes to harvest large numbers of hematopoietic stem cells (HSC) from human placentas both from normal individuals and from patients with Sickle Cell Disease (SCD), using well described markers and functional assays of these cells. In Aim 2, studies will be performed to derive pluripotent, embryonic stem cell (ESC)-like cell lines from placenta. Experiments in the third Aim will seek to develop drug screens to treat SCD, using the HSC isolated from placentas in Aim1.
The research proposed in this application, if successful, has the potential to establish the placenta, a large, readily available human tissue, as a valuable new (and potentially abundant) source of HSC for transplantation applications. The additional possibility of the placenta as a source of cells with pluripotent stem cell-like properties further adds to the potential significance of the proposed studies. Because of the abundance of placenta, there would be little problem in obtaining material from genetically diverse groups. Whether the placenta can be regarded as a truly viable alternative source of either HSC (with more cells per placenta than per cord, for example) or pluripotent human stem cells will critically depend on the results of studies of the nature described in the current proposal.
The PI is an expert in SCD and has a strong interest in generating bankable stores of HSC from placenta. The applicant group has extensive expertise in HSC research and has access to excellent facilities, they will undoubtedly succeed in isolating these cells from placenta. However, one reviewer expressed concern that despite the experience of the PI, a critical marker of the myeloid lineage was incorrectly presented in the experimental plans. Furthermore, reviewers expressed difficulty in evaluating the proposed screens on HSC because of insufficient experimental detail.
The derivation of pluripotent ESC-like cells from placenta is a worthy goal, although a strong focused approach to achieve this goal (as presented for HSC) was not clearly articulated in the proposal. There is strong evidence in the literature for enrichment of stem cell activity in placenta, such as mesenchymal and hematopoietic stem cells, but there is no published evidence for pluripotent stem cells from placenta. In the studies proposed under Aim 2, it is critical that in characterizing the potential pluripotent properties of cells derived from the placenta, comparisons are made with human (h)ESC cells. However, the reviewers were concerned that expertise in hESC biology methodologies is lacking amongst the investigators assembled for this project, and that the proposed experimental design will not allow the applicant to demonstrate pluripotentiality of the placenta-derived cells. Finally, criticism was raised that the drug screening proposed in the third aim does not include the pluripotent stem cells potentially identified in Aim 2.
The idea of deriving pluripotent stem cells from a placental source is responsive to the RFA, although one reviewer was uncertain based on the preliminary data and the proposed experimental plan whether this proposal will yield pluripotent stem cell lines. Furthermore, and more importantly, the majority of this proposal is relevant to generating and using HSC and derivatives. While isolating HSC from placenta is a clinically-relevant and laudable goal, this part of the research plan is not responsive to the RFA.
This proposal is focused on the term human placenta as a high capacity source of stem cells that can be harvested both for therapeutic applications and as a source of cells for drug discovery. The application encompasses three specific aims. In the first, the applicant proposes to harvest hematopoietic stem cells from the placenta using well described markers (CD34, CD133) and to assay these cells in vitro for their colony-forming activity and differentiation potential, and to transplant the cells in NOD/SCID mice to investigate the regenerative capacity of HSC as measured by their ability to initiate and sustain multilineage human hematopoietic cell engraftment. In aim 2, studies will be performed to derive pluripotent (ES-like) cell lines (hPSC) from the chorionic mesenchyme. Cells will be isolated from placenta tissue digests by their adherence to tissue culture plastic/expression of specific cell surface markers typical of pluripotent cells (such as SSEA-3 and TRA-16-0) and morphologic characteristics.
Reviewer One Comments
The placenta is a large, readily available human tissue that has nevertheless been discarded for millennia. The research proposed in this application, if successful has the potential to establish the placenta as a valuable new source of hematopoietic stem cells for transplantation applications much in the way umbilical cord blood was identified as a previously unappreciated source of these cells 20 years ago. The additional potential of the placenta as a source of cells with pluripotent stem cell-like properties further adds to the potential significance of the proposed studies. Whether the placenta can be regarded as a truly viable alternative source of either HSC or pluripotent human cells will critically depend on the results of studies of the nature described in the current proposal.
There are some significant concerns as to the feasibility of the studies proposed under each of the three aims.
In Aim 1, it is not clear based on their track record, whether the PI or any of the other personnel to be involved in this portion of the proposal have any of the necessary expertise or experience in in vitro assays of human hematopoietic cells. Similarly, what experience do they have in the NOD/SCID transplant model, the data included in the preliminary results section notwithstanding? This is of significant concern and speaks to the feasibility of this entire aim. By way of illustration of this concern, the data included under the preliminary results includes mention of engraftment of placenta-derived cells in NOD/SCID mice. While no evidence is provided as to the number of cells infused, the number of mice transplanted, when human cell engraftment was assessed, etc., the greatest concern is that myeloid engraftment is assessed by measurement of CD45+ cells expressing CD25 (the IL-2 receptor). This phenotype in no way measures myeloid engraftment.
In the studies proposed under Aim 2, it is critical that in characterizing the potential pluripotent properties of cells derived from the placenta, comparisons are made with hES cells, a ‘gold standard’ pluripotent cell population. However expertise in ESC biology methodologies is critically lacking amongst the investigators assembled for this project and has not been sought through collaboration. The capacity to establish lines of cells from the placenta exhibiting appropriate cell surface markers that may be maintained following extensive in vitro propagation clearly does not establish these as pluripotent cells. In vitro differentiation assays unless performed at the clonal level are largely meaningless since the applicant cannot exclude the possibility of mixed populations of cells with different potentialities. This caveat applies to the proposed transplant studies in NOD/SCID mice which lack critical detail of experimental design, and will not allow the applicant to demonstrate pluripotentiality of the placenta-derived cells.
The studies proposed under Aim 3 cannot be evaluated. Which compounds will be tested and how many? How will the assays be performed: What endpoints will be measured and how?
The PI Dr Kuypers is a Senior Scientist at the Children’s Hospital Oakland Research Institute (CHORI). His major research interest is in sickle cell anemia and he has a moderate list of publications and has peer reviewed grant funding in this area. A key participant in this proposal is Dr Serikov (80% effort), an Assistant Staff Scientist at CHORI, who brings critical experience in mesenchymal stem cell biology to the proposal.
Responsiveness to RFA:
I cannot ascertain based on the preliminary data and the proposed experimental plan whether this proposal will yield pluripotent stem cell lines.
In regard to plans to share the lines that eventuate from this proposal, the applicant has established extensive collaborative links with academic colleagues at CHORI based on the placenta-derived stem cell populations and with investigators at UC Berkeley, among others. The intention is that by the completion of year 2 the applicant will be able to distribute the placental lines to the broader academic community in California and will utilize a web-based distribution system already established at CHORI (for distribution of cloning vectors, libraries etc.,) for this purpose.
Reviewer Two Comments
The human placenta is a rich source of stem and progenitor cells. If pluripotent stem cells could be isolated from the placenta, this would provide a novel, potentially high yield source of these cells. Because of the abundance of placenta, there would be little problem in obtaining material and cells from very diverse genetic groups, and for banking these cells for potential future therapeutic use. The placenta has been used for the isolation of mesenchymal hematopoietic stem cells in the past. A truly pluripotent population of cells has not yet been isolated from placenta. So though this proposal is only moderately innovative, however, it is highly significant.
The design of the experiments is straightforward. Aim 1 will harvest hematopoietic stem cells from the placenta using traditional methods. The group at CHORI has extensive expertise in hematopoietic stem cell research, including cord blood, and will undoubtedly succeed to isolate these cells from placenta. In Aim 2 the group will attempt to isolate pluripotent stem cells from the placenta using similar methods to Aim 1. The criteria that will be employed to prove pluripotency are naive. Expression of “stemness” markers is not enough. One must keep in mind that none of the stemness markers are unique to pluripotent stem cells. For example, most, if not all of them, are expressed to a high degree on the nullipotent human embryonal carcinoma cell line 2102Ep. The gold standard for human pluripotency has become teratoma formation followed by detailed histological analysis of the tumor. Secondarily, in vitro embryoid body formation is sometimes an acceptable assay. However, directed differentiation and site specific grafting can sometimes yield misleading results, especially when not dealing with a clonal cell population. In Aim 3 the team will demonstrate that the placenta-derived cells can be used in screening assays.
As stated above, the research team seems well suited to accomplishing the proposed studies. They have the required experience. Additionally, the facilities at their disposal are excellent.
Responsiveness to RFA:
This proposal is not responsive to the RFA. In two of the three Specific Aims the researchers focus on hematopoietic stem cells. The work as proposed is fundable by the NIH.
Reviewer Three Comments
This is a project to isolate hematopoietic and pluripotent stem cells from human placentas. This is in principle a very good idea, since there are real advantages in using placenta (large cell numbers, otherwise disposable cells source, patient-specific capabilities, etc.). There is strong evidence in the literature for enrichment of stem cell activity in placenta, in particular mesenchymal stem cells, amniotic epithelial cells, and hematopoietic stem cells. There is no published evidence for pluripotent cells. The PI is expert in sickle cell disease and has a strong interest in generating bankable stores of HSC from placenta. This seems feasible. The derivation of pluripotent ES-like cells is a worthy goal, although there is not a strong focused approach to achieve this goal, as described in the application.
The first Aim is to harvest large numbers of HSC from human placentas, both from normal patients and from SCD. The PI appears to have the resources and expertise to accomplish this goal.
The second aim is to derive pluripotent stem cells. There is relatively little description of how this will be accomplished, other than to look for surface markers such as SSEA3 etc, and then to try and culture FACS-isolated cells into ESC-looking colonies. If successful the lines would be cloned and characterized by morphology, EB and teratoma formation, and in differentiation assays. A rather ambitious list of proposed uses is considered in the context of regenerative medicine, although it is difficult to evaluate these priorities until there is some indication that the lines can be established. No specific genetic modifications or other reprogramming is suggested. Preliminary data indicate that cells positive for Oct4, SSEA3, or Nanog can be identified from placenta.
Experiments in the third Aim will seek to develop drug screens to treat SCD, using the HSC isolated from placentas in Aim1. For this purpose the SCD cells will be useful. The PI will need to efficiently differentiate the HSC to adult stage RBC in vitro. There is not a stated function for the proposed PSC in this Aim.
Responsiveness to RFA:
The idea of deriving PSC from a placental source is interesting and responsive. However, the majority of this proposal is relevant to generating RBC, and testing therapeutic agents on HSC derived from placenta. While an excellent goal, this is not responsive to the RFA, nor is there a strong focused plan for how to derive the PSC in the first place.