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RL1-00640-1: Isolation and Characterization of A Novel Stem Cell Line from Amniotic Fluid and their Potential for Regenerative Medicine Applications

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

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 (provided by applicant)

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

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.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None