Funding opportunities

Cryopreservation of Embryonic Progenitors Derived from Human Embryonic Stem Cells

Funding Type: 
Comprehensive Grant
Grant Number: 
Funds requested: 
$2 088 853
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Efficient cryopreservation and banking of stem cells and stem cell-derived products is a critical component of any comprehensive plan for using these products to regenerate tissues in clinical settings. Banking stem cells and their derivatives provides sufficient time between derivation of cell products and their subsequent distribution to medical centers for safety and efficacy testing to be accomplished. Unfortunately, current methods for cryopreservation of human embryonic stem cells and derivative cell lines are inefficient, hampered by poor recovery, loss of “stem-ness” or self-renewal capability, and changes in gene expression that indicate the nature of the cells has altered during cell processing. Cryopreservation is stressful to cells because it causes rapid changes in water content, formation of dangerous ice crystals, exposure to chemicals that prevent ice crystals but are themselves somewhat toxic, and significant shifts in temperature. Improvements in cryopreservation protocols that minimize cell damage and maintain stem cell characteristics needed for their ability to relocate to and regenerate the corresponding tissues are direly needed. Such improvements will allow the development of an inventory of stem cell products relevant to the wide range of diseases for which stem cell therapy may be applicable. The proposed research will investigate the efficacy of varied cryopreservation protocols on stem cells and populations of stem cell derivatives that appear to represent unique tissues, including some with demonstrated ability to regenerate tissues and restore organ function. Two novel approaches to protect these cells against the stress of cryopreservation will be tested, including the use of a new generation of freezing solutions and a strategy that employs a naturally occurring cell survival signaling pathway known to protect cells against a variety of other biological stress conditions.
Statement of Benefit to California: 
Efficient cryopreservation of stem cell products is a critical component of any comprehensive plan for implementing human tissue regeneration interventions. Improvements in cryopreservation will facilitate banking and safety/efficacy testing of stem cell products relevant to the wide range of diseases for which stem cell therapy may be applicable. The number of Californians who stand to benefit are indicated by yearly mortality rates for coronary heart disease (CHD, 52,000), diabetes-related problems (27,000) and stroke (16,000) and by nonfatal head and spinal cord injury hospitalization rates (25,000 and 1400 per year, respectively). With respect to the tremendous ethnic and racial diversity of California, the impact will be felt across all communities. Hispanics have the highest rate of work-related injuries including burns and trauma. African Americans have the highest rates of CHD and stroke. Pacific Islanders have the highest rates of diabetes-related deaths, followed by African Americans and then Hispanics. Hemoglobinopathies such as sickle cell disease and thalassemia are primarily diseases of African Americans and persons of Asian, Mediterranean and Middle Eastern descent. Craniofacial malformations that affect 1 in 700 children overall have the highest incidence in Native Americans (1 in 300) and Hispanics (1 in 500). Whereas men are by far more likely to suffer head and spinal cord injuries, approximately 12% of American women of childbearing age have sought help for infertility. Improved cryopreservation of gonadal stem cells and oocytes would be a significant benefit to women’s health. As an example of the impact on California’s children, pediatrics burns affect 100, 000 children nationwide per year. The tremendous impact of childhood mortality or disability on society can be better appreciated by measuring years of life and productivity lost. Using this measure, cancer and heart disease still have the highest impact on years lost due to the large numbers of California adults affected by these diseases. However, unintentional injuries and congenital malformations were also ranked highly (3rd and 7th), due to the younger ages of the affected persons. The financial burden of stem cell treatable illnesses is highly significant. For example, in California surgical costs to state service agencies average over $1.5 million per child affected by cleft lip or palate. However, the cost of hospitalizations and surgical treatments does not reflect the indirect toll. For adults, these include costs to industry due to the impact of absenteeism and lost productivity, costs to public programs accountable for their beneficiaries, and other costs to society. In total, the impact of this research is far-reaching with respect to individual lives saved, range of medical conditions, racial and ethnic diversity, women’s health, the state’s economic health and the future generations who will contribute to and enjoy California’s long-term well-being.
Review Summary: 
SYNOPSIS: The proposed project seeks to develop improved methods for cryopreservation of human embryonic stem cells and hESC-derived embryonic progenitors. Efficient cryopreservation and survival after thawing will be important for making hESCs easier to work with and study in general, and in particular facilitate scale up of cultures for differentiation and genetic modification studies. Cryopreservation of hESCs is currently imperfect and is associated with significant cell loss. The first aim of the study is to isolate fresh clonal progenitor cells from hESCs along three germ layer lineages which will be produced by Advanced Cell Technology. Some of these cell lines, which have been derived previously, show cryopreservation inefficiency, thus several progenitor lines will be re-derived and studied in the subsequent aims. Next, the applicants propose to develop rational methods for improving cryopreservation efficiency by testing novel proprietary solutions and using quantitative methodologies to assess a variety of factors. The final aim is to determine whether sphingosine-1-phosphate (S1P) enhances the ability of cells to survive the cryopreservation and thawing process. IMPACT AND SIGNIFICANCE: The proposal is innovative in attempting to isolate and use fresh clonal endodermal, mesodermal, and ectodermal progenitor cell lines from hESCs which have not been formally established, characterized or studied. More likely, the project’s significant impact will be in the area of improved cryopreservation technology. This applied technology development relates to a critical and underfunded area of hESC research whose significance is generally not appreciated. Improving the efficiency of storing hESC lines will facilitate the use of these cells in research and clinical settings because hESC-derived cells will likely need to be stored prior to use in cellular therapies. The cryopreservation of hESC is a problematic technical area, although the efficiency of survival has been significantly improved over the past several years. This work is somewhat high risk because these studies are at an early stage. The PI has expertise in this area, and some preliminary data that supports the second aim of the proposal. The scientific significance of the differentiation studies in the first aim are less clear as they seem to be a repeat of work previously published. Although less well-supported by published data, the use of S1P may further improve the survival of hESC during a freeze-thaw cycle, and the proposal to test novel solutions to protect hESCs from cryopreservation injury may be applicable to many other cell types. Pursuing this type of applied research will speed the translation of hESC research into the clinic. QUALITY OF THE RESEARCH PLAN: The overall quality of the research plan is considered to be strong and the individual aims are well integrated. In Aim 1 the investigators will differentiate hESCs to cells for use in the cryopreservation experiments. The project largely relies on methods previously developed – cells will be provided by ACT. The intellectual innovation of the proposal is in Aim 2 in developing novel cryopreservation protocols. Aim 3 will test whether S1P is an effective cryoprotectant for hESCs and hESC-derived cells. This factor has been shown to improve survival and maintenance of hESC by the Pera lab, and this is a reasonable and easily tested question. The need for developing more cell line samples in Aim 1 is unclear. The clinical utility, characteristics, and usefulness, as well as the purity of the lineage-restricted embryonic progenitor cell types derived from hES cells remains to be seen, and the work in this project could proceed without studies on these particular embryonic cell types, and could be well focused on undifferentiated hESCs themselves. In this case NIH-approved lines could be used and would still lead to valuable new information. The sections of the proposal that aim to improve the survival of hESC during freezing and thawing are well supported, and may yield improved techniques if successful. However, there are three main areas of concern regarding the quality of the research plan. First, a rational assessment of the susceptibility profiles of different cell types in developing a cryoprotectant process (Aim 2) is intellectually satisfying, but unlikely to be effective. The field of cryopreservation is largely empirical. Many labs have attempted to rationally design cryoprotection protocols, but these efforts have not been generalizable to a wide variety of cell types. This does not mean rational design strategies should be abandoned since the potential payoff is very high even though the likelihood of success is low. However, relatively poorly characterized cells, such as hESC derivatives, are not the best choice of cell line for such studies. The methods proposed in this project may improve preservation protocols for a small number of cell types tested but are unlikely to be effective against all or most types. A systematic variation of parameters, though less interesting, would likely provide better results with less effort. Second, the goal of cryopreserving differentiated cells also may be of value, but the preliminary data could be summarized with better quality figures and more complete descriptions. A review of the relevant literature suggests that the samples to be provided by ACT are not in fact cell lines, but heterogeneous populations of differentiated cells that have been characterized up to passage four. While they are derived clonally, they have not been shown to be maintained with a stable phenotype even for these four passages. As sources of differentiated cells, however, they may be useful to answer questions about predicting optimal cryopreservation methods in Aim 2. Third, to develop better cryopreservation protocols the PI has initiated a collaboration with an expert in the field, Dr. Fahy from 21st Century Medicine, and in Aim 2 they will perform a carefully thought out series of studies to test a variety of parameters including osmotic stress and cryoprotectant toxicity. The method described in the proposal as standard, is not in fact a standard cryopreservation method for hESC. Also, the requirement for extensive gene array analysis of cryopreseved samples is not well supported. STRENGHTS: Advances in cryopreservation techniques would be of use to the hESC research community. While these studies are high risk, focusing on the development of general cryopreservation protocols applicable to any cell type rather than testing hESC-specific methodologies enhances the potential impact of the study. This proposal has a well-written and rationally developed research plan which integrates the study of novel survival molecules and new cryopreservation solutions for the storage of human ES cells and their cultured progenitor cell derivatives. This is an ambitious analysis of multiple hESC-derived cell types along with an extensive characterization of the cells following thaw. The PI has extensive experience in the area of cryopreservation, and addresses significant technical challenges of using hESC. In addition, there are good preliminary data indicating the ability to generate hESC-derived clones with distinct transcriptional profiles. The data and publication record indicating the effects of S1P on cell stress responses is also good. Strong collaborators with potentially synergistic expertise are brought together to accomplish the work. The collaboration plan between researchers at Children’s Hospital of Oakland, Advanced Cell Technology, and 21st Century Medicine is well-developed, and as a whole the team is experienced in cell cryopreservation, S1P biology, and hESC biology. WEAKNESSES: The proposal aims to ask technical questions about the cryopreservation of hESC and their differentiated progeny. However, much of the work of the proposal is lacking in clear rationale, for example the need to repeat the extensive differentiation studies and gene expression profiling. Also, it is not clear how the goal of cryopreserving hESC-derived differentiated cells is different from the PI's current NIH-funded projects, except for the origin of the cells. This is a new investigator in the field, without direct experience herself with human ES cell culture or stem cell biology expertise, and there is no description in the proposal of how the PI’s own group will obtain these skills. A major weakness of the proposal lies with the unknown characteristics of the isolated clonal progenitor cell populations. Although Advanced Cell Technology has isolated purportedly clonal progenitor cell lines, the exact characterization of these lines has not been performed. Limited gene expression profiling has been done on them, and in fact it is not clear that the cell lines have the characteristics of true progenitor cells which would include some proliferative capacity, and the ability for differentiation into one or more downstream differentiated cell lineages. It is also not proven that these clonal lines demonstrate purity or a range of cell types along a particular lineage. In addition, no data on endodermal phenotypes is provided. A significant part of the proposal deals with studying the cryopreservation of these progenitor cell lines. As such, this research would have limited applicability if the studies were performed on cells that ultimately proved not to have these progenitor cell characteristics. On the other hand, the main aspect of the proposal deals with simply studying the cryopreservation of undifferentiated human ESCs and this information alone would be useful to the stem cell community if the goals of the proposal were reached. The viability of HEK cells exposed to the novel proprietary cryopreservation solutions demonstrates only a modest twofold improvement compared to DMSO. The key preliminary data on proof-of-concept testing of the novel cryoprotectant solutions on undifferentiated hESCs is not provided; however, if human ESCs and other progenitor cell lines do behave similarly, only slight improvements would be expected to be observed which may not have a significant impact on applications that depend on cryopreservation. Another more minor issue is that 0.5 degrees C/min as a freezing rate may not be optimum. While slower freezing does permit samples to approach equilibrium, faster rates are better for many cell types. A number of key elements in the experimental design are not described. For example, the cell lines to be tested are not indicated in the proposal. The text says they are listed in Figure 2, but they are not, and this information is critical since a reasonable starting point for development of clone-specific methods is an optimized protocol for similar cell types. The specific cryoprotectants to be tested also are not listed. In addition, the length(s) of time in cryostorage and storage conditions are not indicated. Many cell types that are difficult to preserve exhibit substantial viability following short term-freezes but not long-term freezes. Perhaps a less extensive characterization, but performed over multiple samples frozen under different conditions would be more illuminating. Finally, the time point(s) at which post-thaw characterization will be performed is not indicated. The effects of thawing rate, which is as critical as freezing rate for many cell types because cells require a certain amount of time to return to normal gene expression and function, are not discussed. Reviewers highlighted several additional issues. First, the applicants propose to use gene expression profiling to understand the response of the progenitor cell lines to various cryopreservation methods. While it is likely that pre- and post-thaw characterizations by microarrays and gene expression profiling will yield differences, it is not clear what these differences will indicate. Full genome transcriptional profiling is probably not necessary – expression of lineage-specific genes and stress response genes probably would suffice. IHC methods which look at changes at the cellular level would also be informative but these are not proposed. In addition, it is possible that cryopreservation will induce de-differentiation of the progenitor cells in the sense that a progenitor cell state may be relatively unstable. Considerations of what, if anything, the investigators expect to find here are not discussed which reflects a lack of experience in the fields of stem cell and developmental biology. Second, Aim 2 implicitly assumes the mechanism of cell death is related to osmotic shock or cryoprotectant toxicity. In fact, mechanisms including ice formation or apoptosis may dominate for many cell types, including hESCs. In such cases, the proposed methods of developing cryopreservation protocols would be less effective. Third, genetic modification of hESCs to express murine Sphk1 is potentially problematic for multiple reasons. First, such expression will likely alter global transcriptional profiles and differentiation outcomes during the attempted generation of the desired cell clones. Also, genetic modification is undesirable in the generation of cells for human therapies. Direct addition of S1P, proposed as an alternative, would reduce some of these problems as the S1P effects would be temporary, but this method may be less effective. In fact, the rational behind S1P as a potential serum substitute is rather unclear. hESCs viability decreases dramatically in serum-free medium, but the mechanism has not been identified, and enhancement of viability in serum seems a more likely explanation. DISCUSSION: Better cryopreservation methods would be useful for the field, and applying the investigator's expertise in cryopreservation to hESC is a great idea. The applicant is a leader in the field of S1P biology, and the ideas in the proposal are straight-forward with a rational scientific strategy designed to give a clear read-out and yes or no answers. Unfortunately, the remainder of the proposal doesn’t support testing these ideas, and the weaknesses override the positive aspects. For example, there is too little preliminary data on the ACT mixed populations of cells (not lines), and on the key concept of testing for solutions for cryopreservation. The PI has very little expertise in hESC and doesn’t describe culture methods that could affect the success of cryopreservation. In fact, there is no assessment of the quality of the cells before cryopreservation, which is a critical component of the experiments, and reviewers feel that this reflects the PI’s lack of experience with stem cells. Generalizable approaches to improving cryopreservation have been tried for nearly 30 years, and they simply don’t appear to work. Reviewers found parts of the application to be quite valuable and should future funding opportunities arise these should be considered. For example, the sections of the proposal describing the testing of cryopreservation techniques could be separated from the differentiation aims and described in more detail to make a smaller, more focused proposal. In addition, a comparison of the proposed methods with other published hESC cryopreservation methods that are currently in wide use would be useful.

© 2013 California Institute for Regenerative Medicine