Funding opportunities

Isolation and Characterization of A Novel Stem Cell Line from Amniotic Fluid and their Potential for Regenerative Medicine Applications

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00640
Funds requested: 
$1 405 300
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Over the past decade, there has been increasing emphasis placed on stem cells and their potential role in regenerative medicine for the reconstruction of bio-artificial tissues and organs. Embryonic stem cells derived from blastocysts, propagate readily and are capable of forming aggregates (embryoid bodies) that generate a variety of specialized cells including neural, cardiac, and pancreatic cells. However, one of the limitations of embryonic stem cells is their inherent ability to form very complex tumors, known as teratomas, once transplanted into nude mice. From a basic science stand point, teratomas represent primitive embryo development and can serve as a possible model for understanding some of the principles of human tissue differentiation in the laboratory. However, these cells can continue to grow out of control and form tumors. Therefore, teratoma formation is not desirable when developing possible translational or clinical therapies targeted for human applications. In pursuit of safer alternatives, scientists, like myself, have looked at other potential sources for pluripotential cells that can be a viable option to the use instead of embryonic stem cells. Amniotic fluid has been used as a safe and reliable screening tool for genetic and congenital diseases in the fetus for many years. This commonly used diagnostic modality holds very little risk associated with it to either the mother or unborn child. Amniotic fluid also contains a vast repository of progenitor cells that may have a useful role in bioengineering applications. Recently a population of human and rodent amniotic fluid stem cells have been reported in the literature by us and others as giving rise to many different cell types of varying lineages including bone, muscle, fat, and liver with no development of tumors. In our laboratory we have studied extensively this novel population of stem cells derived from amniotic fluid for kidney regeneration. We have demonstrated that this multipotent stem cell population can recapitulate some of the essential steps of kidney development, when injected into an embryonic environment. We would like to continue our efforts in examining this very unique subpopulation of stem cells and validate their role as a true alternative embryonic stem cell line that can be used successfully for regenerative medicine purposes. We plan to characterize fully this new stem cell line and compare them with standard embryonic stem cells to identify important similarities and differences. More importantly, we plan to investigate their ability to ameliorate certain forms of organ diseases, and/or injuries, in an effort to benchmark their potential benefit for reliably safe and biocompatible clinical therapies in the future.
Statement of Benefit to California: 
The State of California is home of some of the nation’s best organ transplant and treatment centers. Having our laboratory next to one of California’s busiest pediatric renal transplant programs in the country {REDACTED} we became acutely aware of the growing shortage of organs for our patients and the need for good alternative therapies in the field of bioengineering and regenerative medicine. The need for donor organs continues to precipitously rise every year and emerging technologies such as those offered by stem cell research may assist our patients and the rest of the citizens in California with alternative technologies that perhaps can make a significant impact in this field. Although human embryonic stem cells show the capacity for multiple differentiation and the prospect for new medical cures, they also have an inherent propensity to form teratomas (benign tumors) in culture which may actually limit their clinical usefulness for future therapies. However, a very novel population of pluripotent stem cells exists within amniotic fluid which can be easily obtained, stored, and possibly used for future regenerative medicine or clinical purposes without the same concerns over clinical compatibility as embryonic stem cells have. These cells could possibly be a safer alternative for regenerative medicine applications aimed at evolving into human clinical therapies that would help those citizens of California in need of organ replacement, or therapies that could ameliorate their conditions. We intend to characterize the potential these cells have for bioengineering applications, a process by which we have extensive experience and success in performing.
Review Summary: 
Executive Summary The stated overall goal of the proposal is to determine the molecular mechanisms and biological evidence for pluripotency of human amniotic fluid stem cells (hAFSC) as compared to an established human embryonic stem cell (hESC) line. Specifically, studies will be performed across three specific aims, the first of which seeks to examine the molecular and cellular mechanisms associated with the pluripotency of AFSC. This will include analysis of hAFSC for gene expression patterns and epigenetic modifications normally associated with the pluripotency of hESC. In addition, the principal investigator (PI) intends to examine the developmental potency of mouse AFSC by introduction into mouse blastocysts and analysis of their capacity to contribute to the development of tissues and organs during development. A second aim focuses on establishing reproducible and robust methodologies to establish, propagate and store hAFSC in a manner that maintains their pluripotent properties and which is compliant with current good manufacturing practices (cGMP). Finally, in a third aim the applicant proposes to investigate the potential of hAFSC to replace damaged tissue in laboratory injury models of lung, kidney, pancreas, and heart. Reviewers concurred that the proposal has potential high significance. Stem cells have recently been isolated from the amnion; however, the pluripotency of these cells has not been extensively evaluated. If successful, derivation of new pluripotent cell lines from amniotic fluid could avoid much of the ethical controversy surrounding hESC lines. Furthermore, once isolated, these stem cells could serve as source material to derive additional lines to cover the genetic diversity of the population. The scope and design of the studies proposed by the applicant encompass, for the most part, the type of analyses necessary to validate some of the key features of the potential pluripotency of AFSC. However, reviewers expressed some general concerns about the feasibility of the proposal. All reviewers criticized that the proposed studies are performed with mixed populations of AFSC. No plan was presented to investigate whether single cell-based clones of AFSC derived from mixed cultures of AFSC exhibit the pluripotent properties of the parental cultures. For one reviewer, this design constitutes a critical flaw, since what appear to be pluripotent properties could represent various limited potentialities of different cell types present in a mixed cell population. Furthermore, reviewers questioned whether the isolation methods and key observations from a publication critical to this work (and cited frequently in the proposal) have been reproduced in the applicant’s laboratory. Reviewers also raised questions about the validity of the proposed pluripotency assays. Directed differentiation in culture, as presented for hAFSC in the preliminary data, can sometimes yield misleading results, especially when not dealing with a clonal cell population. The gold standard for assessing human pluripotency has become teratoma formation followed by detailed histological analysis of the tumor. However, the PI states that AFSC do not form teratomas in SCID mice. Alternatively, in vitro embryoid body formation is sometimes an acceptable assay, but not proposed in this application. Reviewers expressed further feasibility concerns with regard to the very large number of analyses proposed in the second aim, and also felt that the proposed work, although aiming to bank the derived cells, did not adhere to commonly accepted cell banking principles. Under the third aim, a series of experiments is proposed to examine the capacity of AFSC to contribute to the repair of various organ systems (heart, lung, kidney and pancreas) in mouse models. These studies are essential for validating the potential therapeutic applications of AFSC, and are felt to be a strength of the proposal. However, although the PI’s expertise in the kidney and that of a collaborator in the lung speak to the feasibility of the proposed studies in these tissues, reviewers felt that the lack of equivalent expertise for the technically demanding mouse myocardial infarct model raises doubts as to the feasibility of this particular component of the research. Another reviewer was concerned about the absence of rigor in the experimental plan for testing the regenerative properties of these cells, especially in the pancreatic injury model. The PI is an assistant professor based at a research institution which has all the necessary facilities and research infrastructure to support the proposed research. The PI’s previous training was under the mentorship of a noted stem cell researcher with expertise in tissue engineering. The PI has published 19 papers over 10 years (PubMed search). The PI has established important collaborations with a well established investigator in the area of lung development and disease and with an authority in hESC studies. Collectively, the proposed collaborations should ensure that most of the stated aims of the proposal can be achieved. However, this proposal is not completely responsive to the RFA. All reviewers were not convinced that the team will be able to isolate a pluripotent cell population from human amniotic fluid, and therefore, the work as proposed may result in isolation of multipotent stem cells. Reviewer Synopsis The applicant proposes to investigate the properties of a population of cells from human amniotic fluid (termed AFSC). Specifically, studies will be performed across three specific aims, the first of which seeks to examine the molecular mechanisms associated with the pluripotency of AFSC. These proposed studies include global transcriptional profiling to compare the pattern of gene expression with that of established hESC lines, analysis of epigenetic modifications of genes such as Nanog and Oct-4 which are associated with the pluripotency of ES cells, and examination of the developmental potency of AFSC by introduction into mouse blastocysts, and analysis of their capacity to contribute to the development of tissues and organs during development. A second aim focuses on establishing reproducible and robust methodologies to establish, propagate and store AFSC in a manner that maintains their pluripotent properties and which is compliant with cGMP. Finally, in a third aim the applicant proposes to investigate the potential of AFSC to replace damaged tissue in laboratory injury models of lung, kidney, pancreas, and heart models. Reviewer One Comments Significance: Sampling of amniotic fluid has been safely used for many years to provide a source of cells for prenatal diagnosis. Recent observations made by Atala and colleagues have demonstrated the existence within amniotic fluid of a population of cells with extensive proliferative potential and the apparent capacity to differentiate into cells representative of all three germ layers. The research proposed in this application, if successful, has the potential to establish amniotic fluid as a valuable new source of stem cells much as the umbilical cord blood was identified as a previously unappreciated source of hematopoietic stem cells for transplantation. Whether AFSC can be regarded as a truly viable alternative to hESC as a source of stem cells for regenerative medicine applications will critically depend on the results of studies of the kind encompassed by the current proposal. Feasibility: The scope and design of the studies proposed by the applicant encompass, for the most part, the type of analyses necessary to validate some of the key features of the potential pluripotency of AFSC. The preliminary data included in the application appear to attest to the applicant’s ability to isolate AFSC. These data are taken at face value since it is difficult to critically evaluate the observations presented. Figures 5 – 7 which evaluate the effects of transplantation of AFSC in mouse models of renal and lung damage are noted. • AFSC in the present application are to be isolated based on methodologies presumably based upon those reported by De Coppi et al (Nat Biotech 2007). This paper is much cited throughout the application but it would be important to determine if all of the key observations reported in this paper have been reproduced in the applicant’s laboratory and how many AFSC lines with the reported properties have been successfully derived by the applicant and colleagues. • All the proposed studies are performed with mixed populations of AFSC with no attempt being made to investigate whether clones of AFSC derived from mixed cultures of AFSC exhibit the pluripotent properties of the parental cultures. This design constitutes a critical flaw. • Under Aim 1, microarray analysis will be employed to compare patterns of gene expression of AFSC with that of two established hES lines, H1 and H9. The value of these studies is not made clear. There is apparently no intention of comparing gene expression profiles of independent AFSC isolates. How similar are AFSC isolates to each other? What will the applicants conclude if the patterns of expression diverge from each other and from hES cells? • The proposed analysis of the epigenetic state of genes established as critically involved in maintaining pluripotency in ES cells (Nanog, Oct-4, Sox2) is noted. These studies fall short of establishing a functional role for these transcription factors in the regulation of the properties of AFSC. • In the proposed fusion assay, the experimental approach does not make clear how the applicants propose to distinguish between the induction of features of pluripotency in the nucleus of the somatic cell versus those directly contributed by the pluripotent AFSC. • How will the applicants eliminate the possibility that high level contribution of AFSC to multiple adult tissues in the chimera assays is not due to fusion between AFSC and endogenous mouse cells at an early stage of development? • Aim 2 represents an ambitious body of work. The proposed studies are certainly necessary to establish the robustness of AFSC isolation and propagation but multiplying the number of lines by the number of variables to be examined by the number of passages at which these various characteristics will be examined leads to questions of feasibility. • Given the importance of establishing AFSC derivation and maintenance under cGMP compliant conditions, the omission of any attempt to establish AFSC in the absence of FBS is notable. • In the likely event that the AFSC established by the applicants represent a heterogeneous population of cells, what criteria and approaches will be applied to the identification of additional stem cell markers under Aim 1? • Under Aim 3 a series of experiments are proposed to examine the capacity of AFSC to contribute to the repair of various organ systems (heart, lung, kidney and pancreas) in mouse models. These constitute an important series of studies that are essential for validating the potential therapeutic applications of AFSC. However, although Dr DeFilippo’s expertise in the kidney and that of Dr Warburton in the lung speak to the feasibility of the proposed studies in these tissues, the lack of equivalent expertise provided by a collaborator skilled in the technically demanding mouse myocardial infarct model raise doubts as to the feasibility of this particular component of the research. While the intravenous route of AFSC delivery is acknowledged as a potential pitfall, the impact of this route of delivery is perhaps understated and places the outcome of these experiments in the hands of a critical unknown variable, that of biodistribution. The PI Dr DeFilippo is an Assistant Prof in Urology at USC. He is based at the Saban Research Institute of Children’s Hospital in Los Angeles which has all the necessary facilities and research infrastructure to support the proposed research. Dr DeFilippo is a junior investigator whose previous training as a Research Fellow at Children’s Hospital, Boston under the mentorship of Dr Atala furthered his interests in tissue engineering applications in the treatment of renal disease. He has a modest list of publications in peer-reviewed journals. Dr DeFilippo has established collaborations with Dr Wharburton, a well established investigator at Children’s Hospital in LA in the area of lung development and disease and with Dr Pera, an authority in human ES cells recently recruited to the Keck School of Medicine, USC. Both have strong track records in their respective fields of expertise. The contributions of both investigators are vital to the success of the proposed research given the breadth and scope of the proposed studies and the relatively junior standing of the PI. Particularly important is the role of Dr Perin who has direct experience in the isolation and culture of AFSC and is to be employed 50% as a post-doctoral fellow. Collectively, the proposed collaborations should ensure that most of the stated aims of the proposal can be achieved. Responsiveness to RFA: I find it difficult to ascertain based on the preliminary data and the proposed experimental plan whether this proposal will yield pluripotent stem cell lines. AFSC appear to be stable in vitro but whether the purported pluripotent properties are a feature of a single population of cells or of a mixture of cells with various potentialities has not been adequately addressed. The intention of the applicant is to make the AFSC lines widely available once the lines have been quality tested for cGMP manufacturing. Beyond this statement, no more precise plans for distribution are included. I can find no mention in the application of plans to commercialize any lines developed through the proposed research. Reviewer Two Comments Significance: The main goal of this proposal is to isolate and create new cell lines from human amniotic fluid. Such new pluripotent cell lines would avoid the ethical controversy surrounding hESC lines. Once established, it would be trivial to obtain material to derive additional lines to cover the genetic diversity of the population. Stem cells have recently been isolated from the amnion, however, the pluripotency of these cells has not been unequivocally demonstrated. This proposal is moderately innovative and very significant in its approach to the isolation and application of pluripotent stem cells from amniotic fluid. These cells will be used to study mechanisms of pluripotency, as well as potential tools for the repair of damaged and diseased organs. Feasibility: The stated overall goal of the proposal is to determine the molecular mechanisms and biological evidence for pluripotency of amniotic fluid stem cells (AFSC) as compared to an established hESC line. The Specific Aims are logically set forth beginning with the isolation and characterization of pluripotent AFSC (Aim 1), followed by the banking and storage of these cells (Aim 2), and finally the use of the cells to improve recovery after organ injury models (Aim 3). In Aim 1 the PI follows a common misconception. While he is correct in the statement that Nanog, Sox2, and Oct-4 are essential for maintaining ES phenotype, it is not correct to assume that this is what confers pluripotency. Expression of accepted “stemness” genes is not what defines a cell as being pluripotent. One must keep in mind that none of the stemness markers are unique to pluripotent stem cells. Many of these genes are expressed to a high degree in 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. Directed differentiation can sometimes yield misleading results, especially when not dealing with a clonal cell population. The PI states that AFSC do not form teratomas in SCID mice. Another point, the PI needs to be careful not to confuse the term totipotent with pluripotent, as he seems to do occasionally throughout the proposal. In Aim 2 the PI proposes to expand and bank AFSC. He proposes to culture the cells sequentially for 150 population doublings. While this may be good for something, it is not cell banking. Traditional cell banking is designed to avoid the over-expansion of a cell population. It is based on the Master and Working Cell Bank principle and limits the population doublings and passages imposed on the cells. In Aim 3 the PI proposes to use the cells in organ injury models. This Aim appears to be the strength of the team and of the proposal. The PI is particularly accomplished in kidney injury models and brings this experience and know-how to bear. However, one is not convinced that the PI is working with the correct population of cells in this case. The research team possesses the necessary skills and experience to carry out the proposed studies. They have access to suitable facilities. Responsiveness to RFA: This proposal is not completely responsive to the RFA. First, the work as proposed is fundable by the NIH. Next, this reviewer is not convinced that the team will be able to isolate a pluripotent cell population from human amniotic fluid, therefore, the work as proposed will be carried out on a multipotent stem cell at best. Reviewer Three Comments Significance: The main goal is to explore the potential of amniotic fluid derived “stem cells” for regenerative medicine (after tissue injury in lung, kidney, heart and pancreas). A rigorous comparison of these putative stem cells from amniotic fluid to the potential of hES cells is needed, and this project perhaps will accomplish this. Feasibility: There are 3 aims: first, to identify a subpopulation of the amniotic fluid derived cells and to characterize them molecularly and compare to hES cells; second to explore their scale up potential and whether they can form stable lines that can be frozen and reconstituted; third, determine their ability to form replacement cells in various organ injury models. Aim 1 will start with microarray analysis, then analysis of both demethylation and DNA methylation of nanog, sox2 and oct 4 for comparison with “an established” hES cell line even though there is no indication that the population of AFSCs is homogeneous. Another aspect of this aim is to inject mouse AFSC into murine blastocysts to evaluate their ability to integrate into tissue. A concern here is whether the same population can be separated with the same methods from mouse as from human. Aim 2 is a key: it will explore the stability of potential and karotype of 50 c-kit receptor selected samples (to select the AFSC) with repeated expansion and freezing. There is no discussion of the likelihood that only some of the samples will have such stability. Subpopulations will be selected at some point using cell surface markers identified in the microarray gene profiling of aim1. Aim 2 would then determine if true stable cell lines could be derived from these amniotic fluid cells. Aim 3 will use “well described” injury models (that are only briefly described in figure legends of the preliminary data) to test the regenerative capacity of these cells. There are concerns about the rigorousness of testing the regenerative properties of these cells. For example in the pancreatic injury model the use of the beta cell toxin streptotozocin (STZ) is used, but there is no indication of how much damage will be done in these animals. The preliminary data on their beneficial effect suggest the use of low dose STZ model with the cells added just at time of immune response; additionally the cells added seem to have been engineered to express the transcription factor PDX1 to have even this effect. Which model is to be used: One using a multiple low dose STZ model that relies upon a minor toxic effect followed by an immune response, vs. one high dose to destroy significant number of beta cells? Will the AFSC be injected at time of acute injury or once the immune response is evoked? It will be important to distinguish between the AFSC differentiating into beta cells or providing growth factors/anti-inflammatory support to enhance survival of the remaining ones. The PI is an outstanding young physician-scientist who trained with Tony Atala in tissue engineering. He has formed collaborations with several strong scientists (Pera, Warburton) and works with several junior trainees who also trained with Atala. Responsiveness to RFA: The proposal is responsive for the development of the cell lines as well as for their potential in treating specific organ injuries. However, it is still to be shown that these amniotic fluid derived cells can significantly give rise to different cell types (particularly epithelial) either in vivo or in vitro and whether they can be maintained as stable cell lines. Reviewer Four Comments Significance: The proposal calls for the isolation, derivation and characterization of a novel stem cell line derived from amniotic fluid, which is reported to be capable of differentiation into several different lineage/organ types. This proposal will test whether AFSC are equivalent to hESC in pluripotency, although there is already some data from animal models that suggest a lower rate of teratoma formation from AFSC. Feasibility: Design and Feasibility: They will start with an amniocentesis sample, the exact protocol details (such as volume needed) were not defined. They will select for c-kit+ cells (they acknowledge this likely represents a heterogeneous starting cell population, so other additional markers for selection may be necessary, however such candidate other markers were not specified). They will compare genetic, molecular, and cellular properties of AFSC to hESC, using a variety of different assays to determine gene regulatory mechanisms involved in self-renewal, and differentiation potential. Most of this approach was standard, but there were some concerns. They are proposing to perform fusion with somatic cells as a test for ability to reprogram, but it was unclear how these experiments would be interpreted. They are proposing to evaluate the potential for GMP manufacturing of AFSC, but almost no details were provided on this aspect (e.g. FACS sorting to select the starting cell populations is not easily done in a clinically compliant manner) suggesting a lack of understanding of the FDA and the issues involved. There is a whole process of creating Master and Working cell banks for therapeutic use, but in this proposal all that is specifically mentioned is freezing and thawing procedures, testing for stability after 20 cycles of freeze/thaw. The point of this, and its relevance to clinical translation, was unclear. They want to test the potential of AFSC for ameliorating organ injury in various in vivo nude mouse models, and showed some preliminary data suggesting an ability to differentiate into lung, kidney, pancreas. The AFSC were transfected with lac-Z and GFP to allow tracking, and delivered apparently by IV injection. PI has limited funding, for two renal-focused projects: 1) Renal tissue derived from AFSC (no overlap with this proposal, using mouse) and 2) New renal tissue from AFSC (possible overlap with this proposal?) A strength of the application is that they already have SCRO, IRB and IACUC approval Other Concerns: • PI has a limited publication record, and comes from a surgery background. • Limited personnel resources. PI will devote only 10%, Co-investigator is committing only 5%, • Martin Pera listed as consultant, but no % effort. Responsiveness to RFA: It is unclear whether this proposal is completely responsive to the RFA, since it is unclear whether the AFSC are truly pluripotent.
Conflicts: 

© 2013 California Institute for Regenerative Medicine