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RN1-00565-1: Stem Cell Differentiation in Cytopenias
Recommendation: Not recommended for funding
Public Abstract (provided by applicant)
Cytopenias are a major risk factor particularly thrombocytopenia in HIV infection, heart disease, and cancer. Hematopoietic abnormalities such as its inhibition leads to, or cause, multiple cytopenias in HIV infected individuals with thrombocytopenia emerging as a major risk factor for morbidity and mortality and even more so in patients also suffering from heart conditions. Thrombocytopenia is also a major risk factor in cancer patients undergoing chemotherapy. In HIV infected patients, cytopenias are not just caused by infection but are also induced due to prolonged antiretroviral therapy and thus can play a synergistic role. We will investigate herein the mechanisms of inhibition of stem cell differentiation into hematopoietic lineages in vivo.
The mechanisms of hematopoietic inhibition are poorly understood. We use the severe combined immunodeficient mouse cotransplanted with human fetal thymus and liver tissues (SCID-hu) wherein this conjoint hematopoietic organ that develops, to investigate the mechanisms and therapies for inhibition of stem cell differentiation. We have previously shown using this model system that HIV-1 infection inhibits multilineage CD34+ hematopoietic progenitor stem cell differentiation in an indirect manner independent of infection of CD34+ cells in vivo. Furthermore, as a therapeutic strategy this SCID-hu reconstitution model system offers the utilization of either CD34+ cells or even their precursor embryonic stem cells to provide a continuing source of the HSC for alleviation of chronic cytopenias in affected patients. It is hoped that the expected enhancement of stem cell differentiation into multilineage hematopoiesis will aid in decrease of cytopenias in general and thrombocytopenia in particular, in heart disease and cancer therapy.
This model system is also adapted for systemic reconstitution of the stem cells for self-renewal and differentiation to overcome cytopenias. The model will also be tested for the first time to determine if the embryonic stem cell lines such as H1 or H9 will spontaneously or in a directed manner, differentiate into hematopoietic lineages in vivo. In such an event, a greater supply of hematopoietic progenitors will be available to combat cytopenias in vivo. Thus this project involves a combination of mechanistic and therapeutic studies on cytopenias arising from inhibition of stem cell differentiation in vivo.
Statement of Benefit to California (provided by applicant)
The loss of cells in blood (cytopenias) traces their origin to stem cells. This is a general undesirable clinical phenomenon that occurs in various parts of the world including United States either due to natural bodily abnormalities arising from environmental hazards, or from clinical therapies. It has recently been reported [REDACTED] that anemia is widely prevalent [REDACTED] and attributable to environmental factors or causes. Moreover, large cities in California such as San Francisco and Los Angeles have high incidence of HIV infection and both infection and the drugs used in their antiretroviral therapies cause anemia, leucopenia, granulocytopenia and other cytopenias.
Thus for undetermined environmental reasons, the inhabitants of [REDACTED] are vulnerable at least one of the cytopenias, anemia, loss of red blood cells or erythrocytes and hence decreased binding of hemoglobin or availability of iron stores. Cytopenias are a major risk for morbidity and mortality which is particularly the case with thrombocytopenia as it occurs in heart disease. Patients undergoing heart surgery are highly vulnerable to mortality due to thrombocytopenia.
Therefore the studies that we propose are highly beneficial to addressing some of these pressing health issues of California. While certain forms of cytopenias may respond to simpler therapies, it is not clear if the therapy or drug(s) usage can be temporary. Chronic cytopenias may require prolonged or sustained therapeutic interventions and this can be a complicating factor if the patient is also suffering from other clinical conditions. In an extreme case, this complicating factor can even be HIV infection and contributing to greater risk of cytopenias. Or else, it can be a malignancy and that even requires treatment for neutropenia while undergoing autologous stem cell transplantation.
Success in this investigation can produce tremendous benefits to patients suffering from chronic or acute cytopenias and can be life saving in treatments and surgeries or transplantation. Understanding the mechanisms of cytopenias that occur under different conditions will help develop therapies that are tailored to the mode of induction of cytopenias in an individual. We strongly believe that since the origins of cytopenias lie in stem cells so do their therapies.
Review
SYNOPSIS: The overall focus of this grant application is to characterize the cytopenic effects of human immunodeficiency virus (HIV) or HIV-directed drugs as they relate to human hematopoietic progenitor cells. The applicant will use a xenotransplant model to generate chimeric mice engrafted with human fetal liver cells. The investigator will expose these chimeric animals to a large number of drugs that induce cytopenia clinically, and measure the levels of a specific signaling molecule and its ligand. The Principal Investigator (PI) will also test effects on specific signaling pathways. The proposed experiments are based on work of others suggesting a role for a specific factor in hematopoiesis.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: Cytopenia accompanies HIV infection and also is associated with HIV suppressive treatments. This proposal will attempt to understand the possible direct effects of these agents on human hematopoietic precursor cells. In addition, this proposal will attempt to develop a mouse model as a means for expanding autologous hematopoietic stem cells (HSC) for transplant to treat clinical cytopenia; however, there are significant hurdles to the implementation of such a xenograft system, which are not adequately considered.
Unfortunately, the aims of this proposal are very poorly worded so that they almost obscure the larger meaning of the proposal. The proposal suffers from a lack of focus and too much reliance on one assay, which will limit the insights about clinically-relevant issues in stem cell biology in AIDS patients. Additionally, the underlying hypothesis is not well supported by direct evidence and the difficulties of the studies are not recognized, or at least not presented. A major conceptual difficulty is that the in vitro and in vivo studies are not well-correlated to synergize the results. The statistics are presented in isolation with no attachment to specific data sets.
It would certainly be of interest to define (a) the optimal therapy for HIV-1 in terms of least inhibition of stem cell function and (b) a stem cell population that is resistant to HIV-1 infection in the differentiated progenitors. However, the study will not get there as designed. The humanized mouse model is a powerful tool, but many of the studies proposed in the mouse model are better suited to careful in vitro work, and in fact will be cleaner in getting to the core of the issue, which is stem cell toxicity. Much more extensive in vitro work (defining drug and viral effects on stem cell fate) should precede the in vivo studies or at least parallel the animal work.
The proposal aims to generate chimeric mice as a system to study HIV and HIV drug effects on early human progenitor cells. This aspect of the proposal is feasible as the PI has identified reliable tissue sources and the model is up and running in the PI’s lab. However, there are several concerns with the feasibility of the analyses the PI proposes, and the utility of the information that may be gained from them.
The applicant proposes to treat chimeric mice with a large number of drugs both in the presence and absence of HIV infection, alone and in combination. This is a highly ambitious undertaking – no discussion is included of how drug dosage will be determined in this model, which drug combinations will be tested, or how potential toxicity or drug interactions will be controlled for. There is no acknowledgement that the various drugs could have very different effects on the HSC population, and the goal is to determine the mechanism of the inhibition of stem cell function by each drug. A detailed study to get to the heart of this issue (including clonal and lineage assays) would be a huge endeavor, especially if all the drugs turn out to have distinct effects.
The examination of mechanisms of self-renewal is reliant on only two markers, and so much more is known about HSC self-renewal and differentiation. Even if the markers chosen turn out to be relatively good ones for describing the cell fate, they will certainly give an incomplete picture of the toxicity of the drugs to HSC (and viral toxicity).
No clear rationale is provided for the PI’s focus on the specific signaling molecule/ligand and specific signaling pathways in the proposed experiments. Also, it is unclear that the approaches proposed will allow sufficient resolution to discern stem cell vs. progenitor effects. All studies will be done with only partially purified cell populations, which contain both stem and progenitor cells. Also, although aimed at investigating potential self-renewal defects caused by HIV or HIV drugs, the PI does not pose direct experiments to test this and instead relies on surrogate analysis of expression and signaling activity of a specific factor. These signals obviously could change in complex ways that may or may not influence stem cell function. In particular, if regulation of these pathways differs in stem and progenitor cells, which it likely does, then analysis of a mixed population will not be able to distinguish between changes in signaling and changes in population composition.
Support for the “embyronic stem cell phenotype” of marker-positive cells from human fetal liver is lacking. It is unclear what this population of cells represents. If these cells are truly ES-like, then one would expect teratoma formation in immunodeficient animals engrafted by these cells, but the applicant does not consider this potential problem or suggest alternative approaches. This problem arises again in the experiments proposed under a different aim where the PI suggests s/he will “enforce directed differentiation in vivo into hematopoietic stem cells”, but no information is given about how this will be achieved, and no caveats or alternatives are discussed if tumor development occurs.
In general, the proposal lacks the preliminary data needed to support the aims; particularly, demonstration of the ability to differentiate ES cells in vivo to hematopoietic lineages. Achievement of the aims within the proposed timeframe is unrealistic. As presented, the experiments proposed are all very difficult and highly time-consuming, numerous and simply not feasible.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: The PI received a PhD in Biochemistry and has previously trained at several prestigious institutions. The PI obtained his/her first independent position in 2005. The PI’s PhD training has not been in the stem cell or hematology field (it was more focused on biochemistry), and there is some concern that the PI may not yet have mastered this field. The PI lists 18 publications from 1991-2007, with several recent papers published on stem cells and a humanized mouse model. Over the last few years, his/her publication record has not been outstanding despite NIH funding. The enthusiasm of the PI for the proposal is indicated in his/her willingness to devote 50% time to the work.
The applicant outlines a career development plan that focuses first on stem cell differentiation and cytopenia, in line with the focus of the current application. The PI would like to transition his/her research to studying the directed differentiation of hESCs, first to hematopoietic lineages, and then to hepatocytes. The PI would also like to investigate cancer stem cells. Finally, the PI will move back to the hematopoietic system to study leukemia and angiogenesis. In general, this plan touches on multiple important areas of stem cell biology, but lacks focus or explanation for the many different areas the investigator would like to pursue.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The applicant institution is enhancing their stem cell program and recruited the PI as part of this effort. The applicant institution is providing a mentor for the PI, which is a good (and unusual) thing, and the institutional letter of support was much more detailed than most. The PI has his/her own independent lab space, integrated into a strong stem cell group. The institutional environment is well-suited to stem cell research and has a history of promoting and supporting its investigators. The institution supports an on-site vivarium, although this is a very mouse intensive project, and there is some concern that animal space may be limiting. Resources such as confocal microscopy are available off-site at a nearby institution’s core facilities.
DISCUSSION: The PI presents a proposal for a xenotransplant model for cytopenia. Reviewers thought the proposal to be quite disjointed and difficult to read. One reviewer commented that the proposed readouts are problematic as they may not produce meaningful or interpretable results that address the questions of interest. The reviewer also felt that incorrect assumptions were being made about the markers proposed in the study. Overall, there is not much rationale for the experiments proposed. The PI received his/her Ph.D. degree 30 years ago and has only recently (2005) acquired an independent position so s/he does not appear to be a new investigator. Additionally, the PI has not had training in stem cell biology or hematology.
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