Cancer

Coding Dimension ID: 
280
Coding Dimension path name: 
Cancer

Preclinical development of a pan Bcl2 inhibitor for cancer stem cell directed therapy

Funding Type: 
Early Translational II
Grant Number: 
TR2-01789
ICOC Funds Committed: 
$3 341 758
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Cancer Stem Cell
Cell Line Generation: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Cancer is the leading cause of death for individuals under 85. Relapse and metastatic disease are the leading causes of cancer related mortality. Anti-apoptotic BCL2 family member overexpression has been shown to promote disease progression in both chronic myeloid leukemia (CML) and prostate cancer. Andr., the emergence of cancer stem cells (CSC) promotes apoptosis resistance in the bone marrow metastatic microenvironment. While targeted therapy with BCR-ABL inhibitors has improved survival of patients with chronic phase CML, the prevalence has doubled since 2001 with over 22,000 people living with CML in the US in 2009. Unfortunately, a growing proportion of patients become intolerant or simply cannot afford full dose BCR-ABL inhibitor therapy and thus, progress to advanced phase disease with a 5 year survival rate of less than 30%. Although prostate cancer prevalence was high at 2.26 million in 2007, distant disease was relatively rare at 5%. However, like blast crisis CML, metastatic prostate cancer survival was only 30% over 5 years. Overexpression of B-cell lymphoma/leukemia-2 (BCL2) family genes has been observed in human blast crisis CML and advanced prostate cancer and may fuel CSC survival. Recent RNA sequencing data demonstrate that human CSC express a panoply of anti-apoptotic Bcl-2 isoforms in response to extrinsic signals in vivo, indicating that a pan BCL2 inhibitor will be required to abrogate CSC survival. Through binding and anti-tumor studies, a potent inhibitor of BCL2 pro-survival family proteins, BI-97C1, has been identified which inhibits the binding of BH3 peptides to Bcl-XL, Bcl-2, Mcl-1 and Bfl1-1 with nanomolar IC50 values. Notably, BI-97C1 potently inhibits growth of human prostate cancer in a xenograft model as well as blast crisis CML CSC engrafted in RAG2-/-c-/- mice while exerting minimal cytotoxicity toward bax-/-bak-/- cells. Because BI-97C1 inhibits all six anti-apoptotic Bcl-2 family members including Bcl-2, Mcl-1 (myeloid cell leukemia 1), Bcl-XL (BCL2L1), Bfl-1 (BCL-2A1), Bcl-W (BCL2L2) and Bcl-B (BCL2L10) proteins, with improved chemical, plasma and microsomal stability relative to apogossypol, we anticipate that it will have clinical utility for targeting apoptosis resistant human CSC in two malignancies with proven reliance on BCL2 signaling – blast crisis CML and advanced prostate cancer. Thus, anti-apoptotic BCL2 family member inhibition with BI-97C1 could represent a vital component of a potentially curative strategy for advanced malignancies that may obviate the need for costly continuous tyrosine kinase inhibitor therapy by increasing sensitivity to therapy. Elimination of CSC contributing to therapeutic resistance, the primary cause of cancer death, is of high clinical importance and thus, development of a small molecule pan-BCL2 inhibitor would fulfill a vital unmet medical need, fuel California biotechnology stem cell R&D efforts and decrease health care costs for patients with cancer.
Statement of Benefit to California: 
Cancer is the leading cause of death for individuals under 85 and usually results from metastatic disease in the setting of therapeutic recalcitrance. Anti-apoptotic BCL2 family member overexpression has been shown to promote disease progression in both chronic myeloid leukemia and prostate cancer. Moreover, the emergence of quiescent cancer stem cells promotes apoptosis resistance in the bone marrow niche for. While targeted BCR-ABL inhibition has resulted in improved survival of patients with chronic phase CML, the prevalence has doubled since 2001 with over 22,000 people living with CML in the US in 2009 (http://www.leukemia-lymphoma.org). Unfortunately, a growing proportion of patients become intolerant or simply cannot afford full dose BCR-ABL inhibitor therapy as a result of spiraling annual costs and thus, progress to advanced phase disease with a 5 year survival rate of less than 30%. Although prostate cancer prevalence was high at 2.26 million in 2007, distant disease was relatively rare at 5%. Like CML, metastatic prostate cancer survival was only 30% over 5 years (http://seer.cancer.gov/statfacts/html/prost.html#prevalence <http://seer.cancer.gov/statfacts/html/prost.html#prevalence> ). Like blast crisis CML, prostate cancer progression and metastasis is associated with BCL2 overexpression. Thus, anti-apoptotic BCL2 family member inhibition with BI-97C1 could represent a vital component of a potentially curative strategy for advanced malignancies that may obviate the need for costly continuous tyrosine kinase inhibitor therapy by increasing sensitivity to therapy. Elimination of CSC contributing to therapeutic resistance, the primary cause of cancer death, is of high clinical importance and thus, development of a small molecule pan-BCL2 inhibitor would fulfill a vital unmet medical need, fuel California biotechnology stem cell R&D efforts and decrease health care costs for patients with cancer.
Progress Report: 
  • Overexpression of Bcl-2 family genes may fuel CSC survival. Recent RNA sequencing data demonstrate that human CSC express a panoply of antiapoptotic Bcl-2 isoforms in response to extrinsic signals in vivo, indicating that a pan Bcl-2 inhibitor will be required to abrogate CSC survival. Sabutoclax inhibits growth of blast crisis CML CSC engrafted in RAG2-/-c-/- mice with minimal cytotoxicity toward bax-/-bak-/- cells. Because sabutoclax inhibits all six antiapoptotic Bcl-2 family members including Bcl-2, Mcl-1, Bcl-XL, Bfl-1, Bcl-W and Bcl-B proteins, with good chemical, plasma and microsomal stability, we anticipate that it will have clinical utility for targeting apoptosis resistant human CSC in malignancies
  • Significant progress against milestones in the first year was accomplished and we have made early progress on several milestones projected for Year 2. During this 6 month reporting period, sabutoclax was licensed by a biotech company, Oncothyreon. The license was previously held by Coronado Biosciences. Dr. Pellecchia (SBMRI ) continues to provide sabutoclax to Dr. Jamieson for use in cellular and in vivo studies. SBMRI conducted QC analyses (integrity and purity) on samples’ used in preclinical studies and provided comparative analyses of compound produced by the CMO produced by different methods of synthesis. Importantly, the sabutoclax manufacturing process was optimized allowing scale-up of drug. In formulation studies, a method was developed and qualified that separates impurities and degradation compounds from sabutoclax for quantitation of the drug. Additional solubility and stability studies were performed by Oncothyreon to identify an IV formulation that could be used for both nonclinical studies and the clinic. Several pilot PK studies in mice, rats and dogs, planned for Year 2, were also conducted by Oncothyreon. Through whole transcriptome RNA sequencing Dr. Jamieson showed that Bcl-W was up-regulated in CP and BC progenitors compared to normal CB progenitors. Previous qRT-PCR results for Mcl-1 were confirmed, showing that the long isoform was preferentially expressed in BC CML. Results for Bcl-2 and Mcl-1 were also confirmed at the protein level by FACS analysis and immunohistochemistry of bone marrow (BM) from mice engrafted with human CML CD34+ LSC.
  • Sabutoclax treatment ablated BC CML progenitor cells in vivo and in vitro. Colony formation of BC CML (vs normal progenitor cells) was decreased by sabutoclax in a dose dependent manner. When CML cells were co-cultured with stromal cells or in stroma conditioned media, BCL-2 mRNA expression was increased and colony formation was improved. Knockdown of endogenous BCL2 in BC CML cells by shRNA resulted in decreased colony formation. Preliminary results suggest that BM is a protective niche for BC CML CSC and that sabutoclax may target these niche protected cells.
  • In BC CML engrafted mice, dasatinib increased quiescent BC CML cell engraftment in mouse BM measured by FACS for cell cycle markers. Sabutoclax decreased BCL-2 and MCL1 protein expression by immunohistochemistry staining and decreased quiescent BC CML CSC in BM however sabutoclax increased TUNEL staining in BM suggesting that while dasatinib may increase the number of quiescent BC CML CSC, sabutoclax may do the reverse.
  • High doses of sabutoclax administered in combination with dasatinib resulted in a significant decrease in human cell engraftment in BM versus dasatinib alone. Mice serially transplanted with tissues from combination treated mice had increased survival compared to serial transplants of single agent treated tissues. Human CD34+ cells from the BM of combination treated mice had more cells in cycle than CD34+ cells compared to the BM of mice treated with dasatinib alone. The frequency of CD34+BCL2+ and CD34+MCL1+ BC LSC were significantly lower in BM treated with a combination of sabutoclax and dasatinib suggesting that sabutoclax and dasatinib may act synergistically to increase survival of BC CML engrafted mice.
  • Dormant cancer stem cells (CSC) contribute to therapeutic resistance and relapse in chronic myeloid leukemia (CML) and other recalcitrant malignancies. Cumulative data demonstrate that overexpression of BCL2 family pro-survival splice isoforms fuels quiescent CSC survival in human blast crisis (BC) CML. Whole transcriptome RNA sequencing data, apoptosis PCR array and splice isoform specific qRT-PCR demonstrate that human CSC express anti-apoptotic long BCL2 isoforms in response to extrinsic signals in the marrow niche, indicating that a pan BCL2 inhibitor will be required to abrogate CSC survival. Sabutoclax, a novel pan BCL2 inhibitor, prevents survival of BC CSC engrafted in RAG2-/-c-/- mice, commensurate with downregulation of pro-survival BCL2 splice isoforms and proteins, and sensitizes CSC to a BCR-ABL inhibitor, dasatinib, while exerting minimal cytotoxicity toward normal hematopoietic stem cells. Because sabutoclax inhibits all six anti-apoptotic BCL2 family members, with good chemical, plasma and microsomal stability, in addition to a scaleable production process, we anticipate that it will have broad clinical utility for targeting apoptosis resistant quiescent human CSC in a number of recalcitrant malignancies as featured in our recent lead article (Goff D et al, Cell Stem Cell. 2013 Mar 7;12(3):316-28).
  • Significant progress against milestones in the second year was accomplished and we have made early progress on several milestones projected for Year 3. Whole transcriptome RNA sequencing, qRT-PCR array and splice isoform specific qRT-PCR analysis performed on FACS purified progenitors derived from 8 CP, 8 BC and 6 normal samples demonstrated splice isoform switching favoring pro-survival long isoform expression during progression from CP to blast BC CML and in CSC engrafted in the bone marrow (BM) niche. Both human BCL2 and MCL1 protein expression co-localized with engrafted human leukemic CD34+ cells in the bone marrow epiphysis and served as important biomarkers of response to sabutoclax. Importantly, intravenous treatment with sabutoclax reduced BC CML CSC survival in both marrow and splenic niches at doses that spared normal hematopoietic stem cells in RAG2-/-gamma c-/- xenograft models established with cord blood CD34+ cells.
  • While dasatinib treatment alone increased serially transplantable quiescent BC CML CSC in BM, sabutoclax decreased CSC survival commensurate with upregulation of short pro-apoptotic and downregulation of long anti-apopoptotic BCL2 family isoforms. While previous studies involved intraperitoneal administration, in the last 12 months we have focused on a more clinically relevant intravenous (IV) administration schedule with IV sabutoclax administered alone or in combination with oral dasatinib. In these studies, sabutoclax sensitized quiescent CSC to dasatinib resulting in a significant decrease in CSC survival versus dasatinib alone. Moreover, mice serially transplanted with human cells from combination treated mice had increased survival compared to serial transplants of single agent treated tissues. Human CD34+ cells from the BM of combination treated mice had more cells in cycle than CD34+ cells compared to the BM of mice treated with dasatinib alone. The frequency of CD34+BCL2+ and CD34+MCL1+ BC CSC were significantly lower in BM treated with a combination of sabutoclax and dasatinib suggesting that the combination acts synergistically to decrease CSC survival and increase the lifespan of CSC engrafted mice.
  • During this 12-month reporting period, sabutoclax production was successfully scaled up by two separate CMOs, Syncom and Norac. Dr. Pellecchia (SBMRI) provided flash chromatography purified sabutoclax to Dr. Jamieson for use in cellular and in vivo studies in addition to conducting QC analyses (integrity and purity) on scaled up sabutoclax formulations produced by Norac (4g) and Syncom (30g) in different vehicles. In formulation studies, a flash chromatography method was developed and qualified that separates impurities and degradation compounds from sabutoclax. Additional solubility and stability studies were performed to identify an IV Solutol formulation, compared with the previous IP DMSO/PBS Tween formulation, which could be used for both pre-clinical studies and in future clinical trials. Pilot PK studies in mice and rats were conducted with the Solutol formulated sabutoclax and showed weight loss associated with impurities that could be readily removed by standard flash chromatography. As a result, ssabutoclax production will include flash chromatography to enhance purity and stability and this material will be used for further PK and PD studies. In conclusion, we are on track to accomplish our milestones as set forth in the grant and anticipate that sabutoclax will form the basis of combination clinical studies aimed at eradicating quiescent CSC in a broad array of refractory malignancies.
  • Recent cancer stem cell research performed by ourselves and others has bolstered interest in BCL2 family member expression and inhibition in chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and breast cancer (Goff DJ et al Cell Stem Cell 2013; Lagadinou ED et al Cell Stem Cell 2013; Vaillant F et al Cancer Cell 2013). Overexpression of pro-survival BCL2 family genes has been linked to therapeutic resistance driven by dormant, self-renewing CSC. Thus, the BCL2 family represents an attractive therapeutic target that may provide the potential to reduce relapse rates. Because of the greater proclivity for alternative splicing in humans compared with mice, our CIRM ETll funded research has focused on whole transcriptome RNA sequencing, splice isoform specific qRT-PCR and BCL2 PCR array analysis of FACS-purified CSC from patients with CML and CSC derived from human blast crisis CML engrafted RAG2-/-gc-/- mouse models.
  • A Pan-BCL2 inhibitor renders bone-marrow-resident human leukemia stem cells sensitive to tyrosine kinase inhibition. Cell Stem Cell. 2013 Mar 7;12(3):316-28) was featured in a lead article in Cell Stem Cell in March. This study also led to a number of disclosures relating to unique self-renewal and survival gene splice isoform based CSC detection and patient prognostication strategies. As a result, pan BCL2 targeting has generated considerable interest from academic and pharmaceutical investigators who would like to adopt the approach of dormant CSC sensitization to agents that target dividing cells, including tyrosine kinase inhibitors, chemotherapy and radiation therapy.

Forming the Hematopoietic Niche from Human Pluripotent Stem Cells

Funding Type: 
Basic Biology III
Grant Number: 
RB3-05217
ICOC Funds Committed: 
$1 375 983
Disease Focus: 
Blood Cancer
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop. To approach this question we will use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation. Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells. The findings from these studies will have broad applicability to the production of other types of tissues from pluripotent stem cells, all of which have stem cells that require interaction with a specialized niche. In addition to the biological questions explored in this proposal, our focus on the blood system has direct clinical relevance to the field of bone marrow and cord blood transplantation. The development of a human hematopoietic niche from pluripotent stem cells could potentially be used to expand hematopoietic stem cells from adult tissues like cord blood. Most importantly, the ability to control differentiation from pluripotent stem cells into the blood lineage could provide an unlimited source of matched cells for transplantation for patients with leukemia and other diseases of the bone marrow and the immune system who currently lack suitable donors.
Statement of Benefit to California: 
The unique combination of pluripotentiality and unlimited capacity for proliferation has raised the hope that pluripotent stem cells will one day provide an inexhaustible source of tissue for transplantation and regeneration. Diseases that might be treated from such tissues affect millions of Californians and their families. However, much is still to be learned about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The clinical potential of pluripotent stem cells for regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and better controlled than is currently the case. The research proposed in this application has broad potential benefits for Californians both through the biological questions it will answer and the relevance of these studies for clinical translation. Our goal is to understand the way the microenvironment influences tissue production from pluripotent stem cells, a critical issue for the field of stem cell biology. Specifically we will explore the question- Do the cell types produced during differentiation of pluripotent stem cells produce an adequate microenvironment for the differentiation of tissue or are some cells inhibitory to tissue production? Our approach to these questions will be to use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation and offers a range of unique technical tools with which to study these questions rigorously. However, the fundamental concepts formed from these studies will have great relevance for the clinical production of other types of tissues from pluripotent stem cells, such as islets, neural cells and cardiac muscle. In addition to the broad biological questions explored in this proposal, our focus on the blood system has direct clinical relevance to the field of bone marrow and cord blood transplantation. One goal in the proposal is to generate a cellular platform from pluripotent stem cells that will create an environment in which adult blood stem cells can grow and be expanded. Cell numbers collected from cord blood at birth are often insufficient for transplantation in adult patients and older children. The development of a human cell culture system that could expand the number of cord blood stem cells would provide new opportunities for transplantation for patients with leukemia and other diseases of the bone marrow and the immune system who currently lack suitable donors. All scientific findings and technical tools developed in this proposal will be made available to researchers throughout California, under the guidelines from the California Institute of Regenerative Medicine.
Progress Report: 
  • The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop.
  • To approach this question we use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation.
  • Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells.
  • During the first year of support, we have made significant progress in the first two specific aims. We have developed a method that allows us to track the common origin of the blood forming cells and their microenvironment. We also have identified subsets of cells generated from pluripotent cells that have distinct functions in blood formation. Our plan during the next year is to fully characterize these subsets to understand how they function, and to improve our methods to expand them in culture.
  • The clinical potential of pluripotent stem cells for use in regenerative medicine will be realized only when the process by which tissues are generated from these cells is significantly more efficient and controlled than is currently the case. Fundamental questions remain about the mechanisms by which pluripotent stem cells differentiate into mature tissue. The overall goal of this research proposal is to discover if the cell types produced during differentiation of PSC produce the microenvironment needed for specialized tissue stem cells to develop.
  • To approach this question we use the hematopoietic (“blood-forming”) system as our model, as it is the best characterized tissue in terms of differentiation pathways and offers a range of unique technical tools with which to rigorously study questions of differentiation. Adult hematopoietic stem cells (HSC) survive and grow in the bone marrow only if they are physically close to specialized cell types, the so-called hematopoietic stem cell “niche”. We hypothesize that hematopoietic stem cells are not produced from pluripotent cells because the cells that form the niche and provide the necessary signals are not present during this early stage of differentiation.
  • Our research proposal has three specific aims. The first aim is to determine if a single cell type derived from pluripotent cells can generate both blood cells and the cells of the hematopoietic niche. The second aim is to identify the types of niche cells produced from pluripotent cells and define how each of them affect the growth of adult stem cells. In the third aim, the cell types that are found in aim 2 to best support adult hematopoiesis, will then be tested for their ability to promote the production of hematopoietic stem cells from pluripotent stem cells.
  • During the second year of support, we have made significant progress in all three specific aims. We continue to refine our method that allows us to track the common origin of the blood forming cells and their microenvironment during development. We have identified subsets of cells generated from pluripotent cells that can support cord blood HSC and now we are determining the mechanisms by which these cells act and how they can be best used to support HSC that develop from PSC.

The role of neural stem cells in cerebellar development, regeneration and tumorigenesis

Funding Type: 
Research Leadership 1
Grant Number: 
LA1-01747
ICOC Funds Committed: 
$5 919 616
Disease Focus: 
Brain Cancer
Cancer
Cell Line Generation: 
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Stem cells have the remarkable ability to renew themselves and to generate multiple different cell types. This allows them to generate normal tissues during development and to repair tissues following injury, but at the same time, renders them highly susceptible to mutations that can result in cancer. Only by understanding the signals that control growth and differentiation of stem cells can we learn to harness their regenerative capacity and restrain their malignant potential. The research described in this proposal is aimed at elucidating the role of neural stem cells in development, regeneration and tumor formation in the cerebellum. Our previous studies identified a population of neural stem cells in the developing cerebellum. We now propose to use genetic approaches to mark these cells and identify the cell types that they generate during normal development. In addition, we plan to examine the capacity of these cells to regenerate the cerebellum following radiation. Finally, we propose to study the ability of these cells to give rise to brain tumors, and use the models that result from these studies to develop and test novel approaches to therapy. These studies will pave the way towards use of stem cells for repair of neurological damage and help develop more effective treatments for patients with brain tumors.
Statement of Benefit to California: 
We have previously identified a novel population of neural stem cells in the cerebellum. This proposal is focused on understanding the role of these cells in normal development, regeneration and tumor formation. It has the potential to benefit California in a number of important ways. 1. Treatment of Brain Damage: Radiation is the most commonly used treatment for brain tumors, and children who receive this treatment often suffer severe side effects, including a progressive loss of intellectual function. By studying the ability of cerebellar stem cells to repair brain tissue, we will advance the treatment of patients suffering from brain damage due to radiation therapy. The knowledge we gain may also be more broadly applicable, advancing the use of stem cells to repair damage due to congenital brain disorders, trauma and stroke. 2. Treatment of Brain Tumors: Medulloblastoma and astrocytoma are the most common brain tumors in children. By examining the role of stem cells in development of these tumors, we will deepen our understanding of how brain tumors form, and develop novel approaches to treating them. Moreover, we will create new model systems that can be used to test these therapies, with the hope of moving the most effective ones forward towards trials in patients. 3. Technology: Our research will culminate in the invention and generation of new drugs and approaches to therapy that will be made available for licensing by the academic institutions in California, such as {REDACTED} and its collaborators, and developed by pharmaceutical companies based in the State. 4. Collaboration: Our work is multidisciplinary and translational in nature. As such, it will require collaboration with other investigators, including stem cell biologists, neurobiologists, cancer biologists and chemists involved in experimental therapeutics. Once discoveries are made that may be of benefit to patients, we will also work with clinicians to move these discoveries towards the clinic. Californians will be the likely first beneficiaries of these therapies because the clinical trials will be conducted here and we will make an effort to make sure that Californians have immediate access to these therapies when they become standard. By bringing together investigators from various fields and focusing their attention on clinically relevant problems, our studies will advance the translational potential of stem cell research in California.
Progress Report: 
  • The goal of our studies is to determine the role of neural stem cells in the development, regeneration and tumor formation in the cerebellum. By understanding the role of stem cells, we hope to learn to use them for repair of neurological damage and to develop more effective treatments for patients with brain tumors.
  • The aims of our studies are: (1) To identify the cell types generated by cerebellar stem cells during normal development; (2) To determine the capacity of cerebellar stem cells to repair damage caused by radiation or disease; and (3) To determine whether cerebellar stem cells can give rise to the pediatric brain tumor medulloblastoma.
  • We have made significant progress toward our goals over the last year. In particular, we have identified genetic markers that allow us to trace the fate of cerebellar stem cells during normal development. In addition, we have demonstrated that cerebellar stem cells carrying cancer-causing genes can give rise to medulloblastoma. Importantly, this finding has allowed us to create stem cell-based models of medulloblastoma that can be used to test drugs that may be useful for treating the disease. Over the next few years, we hope to use this information to develop more effective therapies for children suffering from medulloblastoma.
  • The goal of our studies is to determine the role of neural stem cells in the development, regeneration and tumor formation in the cerebellum. By understanding the role of stem cells, we hope to learn to use them for repair of neurological damage and to develop more effective treatments for patients with brain tumors.
  • The aims of our studies are: (1) To identify the cell types generated by cerebellar stem cells during normal development; (2) To determine the capacity of cerebellar stem cells to repair damage caused by radiation or disease; and (3) To determine whether cerebellar stem cells can give rise to the pediatric brain tumor medulloblastoma.
  • We have made significant progress toward our goals over the last year. In particular, we have identified genetic markers that allow us to trace the fate of cerebellar stem cells during normal development. In addition, we have demonstrated that cerebellar stem cells carrying cancer-causing genes can give rise to medulloblastoma. Importantly, this finding has allowed us to create stem cell-based models of medulloblastoma that can be used to test drugs that may be useful for treating the disease. Our screening efforts over the past year have begun to identify compounds that inhibit the growth of human medulloblastoma tumor cells. Over the next few years, we hope to use this information to develop more effective therapies for children suffering from medulloblastoma.
  • The goal of our studies is to determine the role of neural stem cells in development, regeneration, and tumor formation in the cerebellum. By understanding the role of stem cells, we hope to learn to use them for repair of neurological damage and to develop more effective treatments for patients with brain tumors.
  • We have made significant progress towards our goals during the past year. We have identified new drugs that potently inhibit the growth of medulloblastoma, the most common malignant brain tumor in children. This work could lead to development of new, more effective therapies for medulloblastoma in patients. In addition, we have developed new models for several types of brain tumors, including one that resembles the most aggressive form of medulloblastoma, and several that model choroid plexus tumors. These models are valuable resources for studying the biology and therapeutic responsiveness of these diseases. Over the next few years, we will continue in our efforts to develop more effective therapies for children suffering from aggressive brain tumors.

Stem Cells in Lung Cancer

Funding Type: 
New Faculty II
Grant Number: 
RN2-00904
ICOC Funds Committed: 
$2 381 572
Disease Focus: 
Lung Cancer
Cancer
Respiratory Disorders
Stem Cell Use: 
Adult Stem Cell
Cancer Stem Cell
oldStatus: 
Active
Public Abstract: 
Lung cancer is the most deadly cancer worldwide and accounts for more deaths than prostate cancer, breast cancer and colon cancer combined. Non small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. The current 5-year survival rate for all stages of NSCLC is only 15%. Although early stage lung cancer has a much better survival rate. Current therapeutic strategies of chemotherapy, radiation therapy and trials with new targeted therapies have only demonstrated, at best, extension in survival by a few months. Clearly, a novel approach is required to develop new therapies for this devastating disease and to detect the disease at an early stage. Cancer stem cells have been identified as the initial cell in the formation of carcinomas. Chemotherapy, radiation and even targeted therapies are all designed to eliminate dividing cells. However, cancer stem cells “hide out” in the quiescent phase of growth. This provides an explanation as to why our cancer therapies may produce an initial response but are often unsuccessful in curing patients. Lung cancer develops through a series of step wise changes that result in the progression of pre-malignant lesions to invasive lung cancer. The mechanisms of how lung cancer develops are not known and if we can prevent the formation of pre-malignant lesions, we will likely be able to prevent lung cancer. We have discovered a subpopulation of stem cells that circulates in the blood and is essential for normal lung repair. Blocking these cells from entering the lung results in a pre-malignant condition in the lungs. We have also identified a subpopulation of stem cells in the lung that is responsible for generating pre-malignant lung cancer lesions. We hypothesize that the interaction between the stem cells in the blood and the stem cells in the lung are critical to prevent lung cancer. We plan to use cutting edge technologies to characterize these different stem cell populations in the lung, and determine how they form pre-malignant lung cancer lesions. We also plan to use preclinical models to try to prevent lung cancer by giving additional stem cells derived from the blood as a therapy. Lastly, we plan to determine whether levels of stem cells in the blood in patients may be used as a blood test to measure the chance of recurrence of lung cancer after therapy. The long term goals of our work are to develop a screening test for lung cancer stem cells that can predict which patients are at high risk for developing lung cancer in order to diagnose lung cancer at an early stage, and to potentially develop a new stem cell based therapy for preventing and treating lung cancer.
Statement of Benefit to California: 
According to the Center for Health Statistics, California Department of Health Services, 13,427 people died of lung cancer in the state of California in 2005. This is more than the deaths attributed to breast, prostate and colon cancers combined. The devastating effects of this disease on the citizens of California and the health care costs involved are enormous. Most cases of lung cancer occur in smokers, but non smokers, people exposed to second hand smoke and ex-smokers are also at risk. In addition, of special concern to California residents, is that exposure to air pollution is associated with an increased risk of lung cancer. Current therapeutic strategies for lung cancer are in general only able to prolong survival by a few months, especially for late stage disease. One reason for this may be that the cancer initiating stem cell is resistant to these therapies. Understanding the stem cell populations involved in repair of the lung and how these cells may give rise to lung cancer is important for potentially generating new therapeutic targets for lung cancer. We propose to study the stem cell populations of the lung that are crucial for normal airway repair and characterize the putative cancer initiating stem cell in the lung. We have also found stem cells in the blood that are critical for normal airway repair and we plan to test their role in the prevention of premalignant lung cancer lesions. We also plan to test whether levels of these stem cells in the blood may be used as a biomarker of lung cancer. Ultimately, the ability to perform a screening test to detect lung cancer at an early stage, and the development of new therapies for lung cancer will be of major benefit to the citizens of California.
Progress Report: 
  • We identified a putative tumor-initiating stem/progenitor cell that goes rise to smoking-associated non small cell lung cancer (NSCLC). We examined 399 NSCLC samples for this tumor-initiating stem/progenitor cell and found that the presence of this cell in the tumor gave rise to a significantly worse prognosis and was associated with metastatic disease. This stem/progenitor cell is known to be important for repair of the airway and is present in precancerous lesions. We believe that this cell undergoes aberrant repair after smoking injury, which leads to lung cancer. We are currently trying to identify the genetic and epigenetic mechanisms involved in this aberrant repair as a means to identify a novel therapy to prevent the development of lung cancer. The presence of these stem/progenitor cells may also be used as a biomarker of poor prognostic NSCLC even in early stage disease.
  • We have identified markers on these stem/progenitor tumor-initiating cells and identified sub-populations of these cells. We are now determining the stem cell capabilities of each of these sub-populations. We are using a model of the development of lung cancer to determine if giving a stem/progenitor cell sub-population for repair can prevent NSCLC from developing.
  • We examined the blood of patients diagnosed with a lung nodule for circulating epithelial stem/progenitor cells. We found that the presence of these cells in the blood of patients predicted the presence of a subtype of NSCLC as compared to a benign lung nodule. We are currently obtaining many more blood samples from patients to further determine whether circulating epithelial stem/progenitor cells could be used as a biomarker of early NSCLC.
  • We have found a stem cell that is important for lung repair after injury that is located in a protected niche in the airway. After repeated injury, for example in smokers, these stem cells persist in an abnormal location on the surface of the airway and replicate and form precancerous areas in the lung. The presence of these stem cells in lung cancer tumors was associated with a poor prognosis with an increased chance of relapse and metastasis.This was especially true in current and former smokers. We therefore believe we have found a putative stem cell that is a tumor initiating cell for lung cancer. We developed a method to isolate these lung stem cells and to profile these cells and developed in vitro and in vivo models to assess their stem cell properties. Finally, we examined human blood samples to assess levels of surrogate markers of these stem cells to assess whether we could use this as a biomarker to predict the presence or absence of lung cancer in patients with a lung nodule.
  • We found a stem cell that is important for lung repair after injury that we believe may form precancerous areas in the lung. We are characterizing these stem cells and identifying pathways involved in normal repair and aberrant repair that leads to lung cancer. We are also isolating this stem cell population and other cell populations from the airway and inducing genetic changes to determine the tumor initiating cell/s for lung cancer. We are also examining the effect the environment may have on the regulation of genes in these stem cells, in precancerous areas and in lung cancers. Finally, we are examining human blood samples to assess levels of surrogate markers of these stem cells to assess whether we could use this as a biomarker to predict the presence or absence of lung cancer in patients with a lung nodule.
  • During this period of funding we discovered a method to reproducibly recover stem cells from human airways and grow them in a dish into mature airway cells. We also discovered the role that certain metabolic cell processes play in regulating the repair after airway injury. We believe that an inability to shut off these processes leads to abnormal repair and lung cancer and are actively investigating this. We are also determining whether the stem cells we isolate from the airways are the stem cells for lung cancer and how they might give rise to lung cancer.
  • In the last year of funding we identified a novel mechanism that tightly controls airway stem cell proliferation for repair after injury. We found that perturbing this pathway results in precancerous lesions that can ultimately lead to lung cancer. Correcting the abnormalities in this pathway that are seen in smokers could allow the development of targeted chemoprevention strategies to prevent the development of precancerous lesions and therefore lung cancer in at risk populations. We also continued our work on trying to identify a cell of origin for squamous lung cancer and identifying the critical drive mutations that are required for squamous lung cancer to develop.

Generation of clinical grade human iPS cells

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00681
ICOC Funds Committed: 
$1 382 400
Disease Focus: 
Amyotrophic Lateral Sclerosis
Neurological Disorders
Melanoma
Cancer
Muscular Dystrophy
Neurological Disorders
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The therapeutic use of stem cells depends on the availability of pluripotent cells that are not limited by technical, ethical or immunological considerations. The goal of this proposal is to develop and bank safe and well-characterized patient-specific pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have the potential to develop into any cell type or tissue type in the body. Importantly, the generation of these cells does not require human embryos or human eggs. Since these cells can be derived directly from patients, they will be genetically identical to the patient, and cannot be rejected by the immune system. This concept opens the door to the generation of patient-specific stem cell lines with unlimited differentiation potential. While the current iPS cell technology enables us now to generate patient-specific stem cells, this technology has not yet been applied to derive disease-specific human stem cell lines for laboratory study. Importantly, these new cells are also not yet suitable for use in transplantation medicine. For example, the current method to make these cells uses retroviruses and genes that could generate tumors or other undesirable mutations in cells derived from iPS cells. Thus, in this proposal, we aim to improve the iPS cell reprogramming method, to make these cells safer for future use in transplant medicine. We will also generate a large number of iPS lines of different genetic or disease backgrounds, to allow us to characterize these cells for function and as targets to study new therapeutic approaches for various diseases. Lastly, we will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases.
Statement of Benefit to California: 
Several groups, including ours have recently shown that adult skin cells can be reprogrammed in the laboratory to create new cells that behave like embryonic stem cells. These new cells, known as induced pluripotent stem (iPS) cells should have, similar to embryonic stem cells, the potential to develop into any cell type or tissue type in the body. This new technology holds great promise for patient-specific stem-cell based therapies, the production of in vitro models for human disease, and is thought to provide the opportunity to perform experiments in human cells that were not previously possible, such as screening for compounds that inhibit or reverse disease progression. The advantage of using iPS cells for transplantation medicine would be that the patient’s own cells would be reprogrammed to an embryonic stem cell state and therefore, when transplanted back into the patient, the cells would not be attacked and destroyed by the body's immune system. Importantly, these new cells are not yet suitable for use in transplantation medicine or studies of human diseases, as their derivation results in permanent genetic changes, and their differentiation potential has not been fully studied. The goal of this proposal is to develop and bank genetically unmodified and well-characterized iPS cell lines of different genetic or disease backgrounds that can be used to characterize these cells for function and as targets to study new therapeutic approaches for various human diseases. We will establish protocols that would allow the preparation of these types of cells for clinical use by physicians investigating new stem cell-based therapies in a wide variety of diseases. Taken together, this would be beneficial to the people of California as tens of millions of Americans suffer from diseases and injuries that could benefit from such research. Californians will also benefit greatly as these studies should speed the transition of iPS cells to clinical use, allowing faster development of stem cell-based therapies.
Progress Report: 
  • The goal of this project is to develop and bank safe, well-characterized pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases, and that are not limited by technical, ethical or immunological considerations. To that end, we proposed to establish protocols for generation of human induced pluripotent stem cells (hiPSC) that would not involve viral vector integration, and that would be compatible with Good Manufacturing Processes (GMP) standards. To establish baseline characteristics of hiPSCs, we performed a complete molecular characterization of all existing hiPSCs in comparison to human embryonic stem cells (hESCs). We found that all hiPSC lines created to date, regardless of the method by which they were reprogrammed, shared a common gene expression signature, distinct from that of hESCs. The functional role of this gene expression signature is still unclear, but any lines that are generated under the guise of this grant will be subjected to a similar analysis to set the framework by which these new lines are functionally characterized. Our efforts to develop new strategies for the production of safe iPS cells have yielded many new cell lines generated by various techniques, all of which are safer than the standard retroviral protocol. We are currently expanding many of the hiPSCs lines generated and will soon demonstrate whether their gene expression profile, differentiation capability, and genomic stability make them suitable for banking in our iPSC core facility. Once fully characterized, these cells will be available from our bank for other investigators.
  • For hiPSC technology to be useful clinically, the procedures to derive these cells must be robust enough that iPSC can be obtained from the majority of donors. To determine the versatility of generation of iPS cells, we have now derived hiPSCs from commercially obtained fibroblasts derived from people of different ages (newborn through 66 years old) as well as from different races (Caucasian and mixed race). We are currently evaluating medium preparations that will be suitable for GMP-level use. Future work will ascertain the best current system for obtaining hiPSC, and establish GMP-compliant methodologies.
  • The goal of this project is to develop and bank safe, well-characterized pluripotent stem cell lines that can be used to study and potentially ameliorate human diseases. To speed this process, we are taking approaches that are not limited by technical, ethical or immunological considerations. We are establishing protocols for generation of human induced pluripotent stem cells (hiPSCs) that would not involve viral vector integration, and that are compatible with Good Manufacturing Practices (GMP) standards. Our efforts to develop new strategies for the production of safe hiPSC have yielded many new cell lines generated by various techniques. We are characterizing these lines molecularly, and have found hiPSCs can be made that are nearly indistinguishable from human embryonic stem cells (hESC). We have also recently found in all the hiPSCs generated from female fibroblasts, none reactivated the X chromosome. This finding has opened a new frontier in the study and potential treatment of X-linked diseases. We are currently optimizing protocols to generate hiPSC lines that are derived, reprogrammed and differentiated in the absence of animal cell products, and preparing detailed standard operating procedures that will ready this technology for clinical utility.
  • This project was designed to generate protocols whereby human induced pluripotent stem cells could be generated in a manner consistent with use in clinical trials. This required optimization of protocols and generation of standard operating procedures such that animal products were not involved in generation and growth of the cells. We have successfully identified such a protocol as a resource to facilitate widespread adoption of these practices.

Mechanisms to maintain the self-renewal and genetic stability of human embryonic stem cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00148
ICOC Funds Committed: 
$2 570 000
Disease Focus: 
Cancer
Genetic Disorder
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Human embryonic stem cells (hESCs) are capable of unlimited self-renewal, a process to reproduce self, and retain the ability to differentiate into all cell types in the body. Therefore, hESCs hold great promise for human cell and tissue replacement therapy. Because DNA damage occurs during normal cellular proliferation and can cause DNA mutations leading to genetic instability, it is critical to elucidate the mechanisms that maintain genetic stability during self-renewal. This is the overall goal of this proposal. Based on our recent findings, I propose to investigate two major mechanisms that might be important to maintain genetic stability in hESCs. First, I propose to elucidate pathways that promote efficient DNA repair in hESCs. Second, based on our recent findings, I hypothesize that another primary mechanism to maintain genetic stability in self-renewing hESCs is to eliminate DNA-damaged hESCs by inducing their differentiation. Therefore, I propose to identify the pathways that regulate the self-renewing capability of hESCs in the presence and absence of DNA damage. In summary, the proposed research will contribute significantly to our understanding of the pathways important to maintain self-renewal and genetic stability in hESCs. This information will provide the foundation to improve the culturing condition of hESCs to promote efficient self-renewal with minimum genetic instability, a prerequisite for the development of hESCs into human therapeutics. One major objective of the proposed research is to improve the genetic manipulation technologies in hESCs, including transgenic and gene targeting technologies. While mouse models are valuable tools to study the mechanisms of the pathogenesis in human diseases, many differences between mouse and human cells can lead to distinct phenotypes as well as the common phenomenon that certain therapeutic interventions work well in mouse models but poorly in humans. Therefore, it is of high priority to create disease-specific hESCs as powerful genetic tools to study the mechanism of the pathogenesis in human diseases. In addition, the unlimited supply of primary cells derived from the disease-specific hESCs will become valuable reagents for drug discovery. There are two ways to generate the disease-specific hESCs. One approach is through nuclear transfer that has been proven extremely difficult in human context and so far unsuccessful. The other is to employ the transgenic and gene targeting techniques to create disease-specific hESCs. Therefore, the proposed research will significantly improve our capability to generate disease-specific hESCs. After experimenting with various existing hESC lines, we found that only the non-federally-approved hESC lines developed recently at Harvard University is most suitable for genetic manipulation technologies. Since the research involving the HUES lines can not be supported by federal government, CIRM is in a unique position to support this proposed research.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) are capable of unlimited self-renewal, a process to reproduce self, and retain the ability to differentiate into all cell types in the body. Therefore, hESCs hold great promise for human cell and tissue replacement therapy. The major goal of the human stem cell research supported by proposition 71 is to improve and even realize the therapeutic potential of hESCs. DNA damage occurs during normal cellular proliferation of hESCs and can cause genetic mutations that will be passaged to derivatives. Any cells with genetic mutations are not suitable for therapeutic purpose since they can cause cancers in the recipient. Therefore, to achieve the therapeutic potential of hESCs, it is critical to elucidate the mechanisms that prevent genetic mutations during the self-renewal of hESCs. This is the overall goal of this proposal. Successful completion of the proposed research will help to optimize the culturing conditions that promotes efficient self-renewal with minimum genetic instability. One high-priority area of hESC research is to create disease-specific hESCs, which can be used as powerful genetic tools to study the mechanism of the pathogenesis in human diseases. In addition, the unlimited supply of primary cells derived from the disease-specific hESCs will become valuable reagents for drug discovery. There are two ways to generate the disease-specific hESCs. One approach is through nuclear transfer that has been proven extremely difficult in human context and so far unsuccessful. The other is to develop the transgenic and gene targeting techniques to create disease-specific hESCs. One major objective of my proposed research is to improve the genetic manipulation technologies in hESCs, including transgenic and gene targeting technologies. The successful completion of the proposed research will significantly improve our capability to generate disease-specific hESCs. In addition, the disease-specific hESCs (ATM-/- and p53-/- hESCs) generated in the course of the proposed studies are valuable tools to study the basis of neuronal degeneration in Ataxia-telangiectsia and development of human epithelial tumors as a result of p53-deficiency. Both of these phenotypes are not observed in mouse models. In summary, the proposed research will benefit California citizens by contributing to the eventual realization of the therapeutic potential of hESCs.
Progress Report: 
  • The goal of this proposal is to investigate the mechanisms that maintain the genomic stability of human ES cells (hESCs). We are focusing on the tumor suppression pathways ATM and p53, which are well established guardians of the genome in differentiated cells. In addition, we are investigating the pathways that govern the self-renewal of hESCs, which might be coordinated with DNA damage responses to maintain the genomic stability in hESCs. During the reporting period, we made significant progress towards our goals. First, we developed high efficiency homologous recombination technology to successfully disrupted ATM and p53 in hESCs. Analysis of the mutant ES cells indicate the roles of ATM and p53 in maintaining genomic stability in hESCs. Second, we identified pathways that are important for the self-renewal of hESCs. Third, we employed the knock-in tech
  • The goal of this proposal is to investigate the mechanisms that maintain the genomic stability of human ES cells (hESCs). We are focusing on the tumor suppression pathways ATM and p53, which are well established guardians of the genome in differentiated cells. In addition, we are investigating the pathways that govern the self-renewal of hESCs, which might be coordinated with DNA damage responses to maintain the genomic stability in hESCs. During the reporting period, we made significant progress towards our goals. First, we developed a bacterial artificial chromosome based gene targeting technology that allows high efficiency homologous recombination in hESCs, and published the first homozygous knockout mutant hESCs in the world (Aims 1 and 3). This achievement, which was described in a publication in the top stem cell journal Cell Stem Cell, has attracted worldwide attention and will help to open up the entire field of hESCs (Song et al., 2010, Cell Stem Cell 6, 180-189). We employed the same technology to generate homozygous phosphorylation site knock-in mutant hESCs to study the mechanism underlying ATM activation in hESCs (Aim 3). Second, we identified a novel Pin1-Nanog pathway that is critical for the self-renewal of hESCs (Aim 2). Using small molecule compounds that inhibit this pathway, we were able to suppress the potential of ES cells to form teratomas. This finding, which is published in the Proceeding National Academy of Science, provides a druggable target to address the teratomas risk associated with the human ES cell based therapy (Moretto-Zita et al., 2010, PNAS, Epub 7/9). Third, to identify ES cell-specific DNA repair pathways, we have identified several ES cell-specific interaction between proteins and DNA breaks (Aim 3).
  • We have made several significant progresses during the past year. We found the important roles of p53 in the differentiation of hESCs. We also identified that Nanog is a major coordinator of the self-renewal and proliferation of ES cells. We found that ATM is important to maintain the genetic stability of cells differentiated from hESCs. In addition, we identified an important phosphorylation event in activating ATM in hESCs. Finally, we identified a novel pathway to activate DNA damage in ES cells.

Role of the tumor suppressor gene, p16INK4a, in regulating stem cell phenotypes in embryonic stem cells and human epithelial cells.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00444
ICOC Funds Committed: 
$639 150
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The roles of stem cells are to generate the organs of the body during development and to stand ready to repair those organs through repopulation after injury. In some cases these properties are not correctly regulated and cells with stem cell properties expand in number. Recent work is demonstrating that the genes that control stem cell properties are sometimes the same genes that are mutated in cancer. This means that a cell can simultaneously acquire stem cell properties and cancer properties. In order to effectively use stem cells for therapeutic purposes we need to understand the link between these two programs and devise ways to access one program without turning on the other. In other words, we would like to expand stem cell populations without them turning into cancer. Recent work in our laboratory has found that the reduction of a specific tumor suppressor gene, p16, not only removes an important barrier to cancer but also confers stem cell properties within the cell. Cells that have reduced p16 activity can turn on a program that increases and reduces expression of specific genes that control differentiation. In this proposal we will test whether the continued reduction of this tumor suppressor gene creates human embryonic stem cells (hESC) that are unable to differentiate. We hypothesize that the lack of p16 represses multi-lineage potential by activating an epigenetic program and silencing genes that drive differentiation. To test this hypothesis we will first determine if lack of p16 activity is necessary for hESCs to develop into different cell types. Second, we will determine if continued lack of p16 activity is sufficient to inhibit differentiation of hESCs. Finally, we will determine if transient lack of p16 activity is sufficient for a non-stem cell to exhibit properties of a stem cell after propagation in a stem cell niche. Since these types of events are potentially reversible, targeting such events may become clinically useful. These new observations identify novel opportunities. They provide potential markers for determining if someone is susceptible to cancer, as well as, providing potential targets for prevention and therapy. We hypothesize that these properties are critically relevant to the formation of cancer and will provide insights into the role of epigenetic modifications in disease processes and stem cell characteristics.
Statement of Benefit to California: 
Stem cells hold great potential to help us in repairing injured body parts or replacing damaged organs. In order to realize this potential the rules that control stem cell behavior need to be understood. Recent work is demonstrating that the genes that control stem cell properties are sometimes the same genes that are mutated in cancer. In the proposed study we hypothesize that we may learn about a fundamental switch that not only controls stem cells but also controls the formation of a cancer cell. In understanding how this switch works we may be able to identify biomarkers that indicate when a normal looking cell will become a cancer cell or identify a drug that will allow us to stop the potential cancer cell from increasing in number. Since cancer is a common disease in California, any insights we can gain to battle this disease will benefit the citizens of our State. There is also another side to the insights that may arise from the work in this proposal. Currently we believe the roles of stem cells are to generate the organs of the body during development and to stand ready to repair those organs through repopulation after injury. We do not know how to encourage a stem cell to repair, for example, some heart tissue rather than some bone tissue. If we could understand the code that directs the stem cells to differentiate in the proper fashion into one tissue or another, we could use these cells for clinical benefit. The pathways we are studying in this proposal tell the stem cells which genes to silence and which to activate. This is the program that allows the one original cells of your body (the embryo) to diversify into the multitude of specialized cells that work together to make a functioning person (eye cells, skin cells, nerve cells, etc.). In order to effectively use stem cells for therapeutic purposes we need to understand how they code their decisions and whether they can be changed after they have been set. These insights would allow us to aid in maintaining the health of the citizens of California. Finally, if we do gain insight into the code that regulates the differences between cancer cells and stem cells, this information would be the basis of a new area of biotechnology. The generation of knowledge in this area would help in the development of companies, the recruitment of bright young minds and in the fiscal health of our State
Progress Report: 
  • Stem cells hold great potential to help us in repairing injured body parts or replacing damaged organs. In order to realize this potential the rules that control stem cell behavior need to be understood. Our laboratory has found that repression of the tumor suppressor p16 in human mammary epithelial cells (HMECs) endows them with specific properties that are only found in classical stem cells and tumor cells. Indeed, repression of p16INK4a in HMECs enables them to grow in culture for a long time, something that HMECs expressing p16INK4a cannot achieve. Importantly, we have previously shown that repression of p16INK4a is accompanied by the acquisition of pre-malignant features.
  • Thanks to the support of this CIRM grant, we have now established that a sub-population of these cells display stem cell properties. This means that these cells can self-renew but also differentiate in different breast cell types. Unexpectedly, these cells can also give rise to non-breast cells, such as brain cells, when grown in the appropriate cell culture conditions, making this unique cell model a powerful tool for cancer AND regenerative medicine research. Knowing that these cells can generate cells of different tissue types, we can now dissect the rules that dictate those different cell fates. We are also testing whether these exciting findings obtained in cell culture dishes (in vitro) can be confirmed in a mouse model (in vivo). In other words, can these cells generate a functional mammary gland? Other studies, beyond the scope of this application could also test whether these cells could rescue spinal injury.
  • So why do we bother using breast cells to generate brain cells (or other types of cells)? The answer is that we believe that the sub-population of cells we have identified in breast likely exists as a stem cell pool in any tissue (with some tissue-specific variations of course). If this hypothesis is confirmed, these cells could turn out to represent a major advancement in regenerative medicine. Another major advantage of these naturally occurring stem cells, compared to the widely used embryonic stem cell lines, is that they are directly isolated from fresh breast tissue without introducing artifacts that may result from establishment in long-term cell culture systems. Their properties are an accurate reflection of a fully functional stem cell pool actually existing physiologically in our body.
  • Understanding how stem cells code their decisions and whether cell fate can be changed after it has been set is key to the effective use of stem cells for therapeutic purposes. Gaining such insights will greatly improve our ability to manage wound repair and organ replacement. This should also help us characterize fundamental switches that control stem cells as well as control the formation of cancer cells since some of the genes that control stem cell properties are mutated in cancer. A mechanistic understanding of how these switches work may help us prevent adverse events that may result from the use of stem cells during regenerative medicine. Thus, we hope to contribute in improving the health of the citizens of California.
  • An important feature of adult stem cells is the ability to bypass negative growth signals and participate in wound healing. Based on this premise, we identified a small subpopulation of human breast epithelial cells that is capable of bypassing negative growth signals. We identified a differential expression of genes that allowed for the rapid isolation of this novel somatic cell population from fresh disease-free human breast tissue. Importantly, this cell population is characterized by the over-expression of Bmi-1, a protein that plays an essential role in the self-renewal of stem cells and represses the cell cycle inhibitor, p16. This population of cells is therefore poised to express pluripotency markers at a level similar to that measured in human embryonic stem cells. It has the ability to self-renew and can express phenotypes of any of the three mammary lineages in vitro using cell culture differentiation assays. Importantly, these cells are also functional in vivo as observed after implantation in mice. Indeed, these human cells can differentiate into functional mammary outgrowths of human origin in the host mouse as we could document secretion of human milk in mice transplanted with these human somatic cells. We are currently investigating whether these cells can also differentiate into other lineages (tissue types) when cultured in the appropriate conditions. Our preliminary studies support that these cells will hold great promise in regenerative medicine and cell replacement therapy and may help overcome some of the important ethical and technical roadblocks related to the use of human embryonic stem cells.

Sources of Genetic Instability in Human Embryonic Stem Cells.

Funding Type: 
SEED Grant
Grant Number: 
RS1-00428
ICOC Funds Committed: 
$357 978
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The constant exposure of cells to endogenous and exogenous agents that inflict DNA damage requires active repair processes to eliminate potentially mutagenic events in stem cells leading to cancer. The same agents menace early human embryos with DNA damage that can ultimately lead to mutations, cancer, and birth defects. In vitro, human embryonic stem cells (HESCs) spontaneously undergo events leading to genetic instability and mutations. All these three types of genetic problems can have similar links to malfunctions in DNA repair systems, but little information now exists for HESCs. Therefore, the first step in understanding the causes of HESC genetic instability is to understand which DNA repair systems are defective. We will investigate the basis for this phenomenon in HESCs by evaluating their capacity to either repair DNA or form mutations. First, we will culture two HESC lines and compare HESC repair and mutation formation to that of control cells. We will use a new technique which simplifies the production and use of the feeder cells that support the growth of the HESCs. We will also test the genetic stability of HESCs grown on conventional feeder cells, as well as those grown in feeder free culture. We will use three types of DNA repair assays to monitor the genetic stability of the two HESC lines grown in these different ways. In the first of these assays, DNA molecules with different randomly-induced damage are transferred into HESCs, and DNA repair is followed by the re-establishment of the activity of a reporter protein that is coded for in the damaged DNA. A second assay will introduce specific DNA damage at a unique site in DNA that is transferred to HESCs and repair is determined using a polymerase chain reaction-based technique. Since aneuploidy is also known to be caused by double-strand DNA breaks, we will use two other assays to evaluate capacity of HESCs to repair that type of damage. These experiments will indicate if DNA repair pathways that eliminate DNA damage are dysfunctional and cause genetic instability. The final endpoint for these preliminary experiments is the formation of mutations. To study this, we have modified an assay system so that it will function in normal human cells to monitor mutations which arise spontaneously or those which are induced by various agents. In summary, these investigations will provide the basis for understanding genetic instability in HESCs that can direct cells to tumorgenic outcomes. The employment of HESCs clinically will require such knowledge. Moreover, these results will also yield information on susceptibility to mutations of cells early in development. The practical and basic science aspects of this seed grant proposal should lead to a complete proposal in the near future.
Statement of Benefit to California: 
Human embryonic stem cells (HESCs) hold the potential to cure or alleviate many chronic illnesses, including cancer, but an immense gap exists between the achievement of the goals of stem cell based medicine and the current state of the art. Several stages of development including the following are required: (1) Routine, standardized, simple protocols for the indefinite growth of HESC in the normal, undifferentiated state, in completely defined medium.(2) Control over differentiation of the cells in (1) to all adult cell types of interest. (3) Control over the maintenance of the differentiated state of derivatives of (1), in sufficient complexity to recreate normal functional histology. (4) Techniques, therapies, and protocols that allow immune tolerance of regenerated tissue, without rendering the human recipient immunodefficient. Researchers are still struggling with steps 1 and 2. Although claims of feeder cell and animal product-free, long-term, undifferentiated HESC culture have been made, this is not the current state of the art in laboratories. These claims may be fortuitous or true for only a few HESC lines. The public anticipates a quick success of human stem cell technology and application to human disease, but the promise of stem cell therapy requires basic scientific work that is critical, but may not make headlines. Imprudent claims of miraculous cures could dim public enthusiasm. Few if any data exist regarding DNA repair systems or mutation frequencies of HESCs. We propose to investigate mechanisms underlying genetic instability in cultured HESCs. This instability limits HESC research and therapeutic applications. The data generated by this research according to Proposition 71 will be of lasting value to the People of the State of California for the following reasons: (1) This proposal focuses on a serious, basic difficulty with respect to the growth of undifferentiated HESCs that is a barrier to their human therapeutic use.(2) In the future, if the focus of the stem cell field shifts to the as yet unavailable somatic nuclear transfer (SNT) methods, this proposed research, will provide a basis for the comparison of HESCs and SNT cell lines. (3) All humans begin as embryonic stem cells, therefore data generated by the proposed research will impact maternal health, well baby programs, early childhood development/learning, etc, because mutations are involved in birth defects as well as cancer. Therefore, understanding the causes of mutations in HESCs could assist in avoidance or reduction in birth defects that would aid both the families and the government of California.(5) All of the work described in this proposal will be conducted by individuals in California and most probably will result in the hiring of a graduate of a California institution of higher education, thus reducing unemployment and helping educate a new generation of California researchers in HESC use.
Progress Report: 
  • Human embryonic stem cells (hESCs) originate directly from human embryos, whereas induced pluripotent stem cells (iPSCs) originate from body (somatic) cells that are re-programmed by producing or introducing proteins that control the process making specific RNAs. Together, both these pluripotent cell types are referred to as human pluripotent stem cells (hPSCs). Several reports have observed that in hESCs grown for long times, their genetic material, DNA, is unstable. The stable maintenance of DNA is performed by groups of proteins functioning in different systems globally known as DNA repair pathways. Since the development of aneuploidy is closely linked to cancer and to deficiencies in DNA repair, we have studied the propensity of hPSCs to repair their DNA efficiently by 4 major known DNA repair pathways. In addition, we are also investigating if specific damage to DNA in either hPSCs or somatic cells is processed differently and could lead to deleterious mutations.
  • One major goal of the CIRM SEED grant mission is to bring new researchers into the hPSC field. The results we obtained during the funding period indicate that we have succeeded in that objective, since initially our laboratory had little experience with hESC culture. However, through courses and establishing critical collaborations with other hESC laboratories, we developed expertise in hPSC culture techniques. Most conditions for hPSCs growth require cells (feeder cells) that serve as a matrix and provide some factors needed for the pluripotent cells to divide. In accomplishing this aim, we perfected a method to generate reproducible feeder cells that significantly reduces the time and cost of feeder cell maintenance, and also developed a non-enzymatic and non-mechanical way to expand hPSCs. We now have experience with at least 5 hPSC lines and have methods to introduce foreign DNAs into hESCs and iPSCs to monitor DNA repair in hPSCs.
  • In Aim II of our grant, we used our accumulated knowledge of hPSCs and DNA repair to investigate 4 DNA repair mechanisms in hPSCs and in somatic cells. Depending on the DNA damage, there is often a preferred DNA repair pathway that cells use to alleviate potential harm. We initiated our investigation by treating hPSCs using different DNA damaging agents, including ultraviolet light and gamma radiation. However, we found that hPSCs exposed to these agents rapidly died compared to treatments that allowed somatic cells to continue growing. Therefore, we developed methods to study DNA repair in hPSCs without directly treating the cells with external agents. We treated closed, circular DNA (plasmids) with damaging agents separately, outside the hPSCs and then introduced them into the hPSCs. The plasmid DNA has a sequence that codes for a protein that is produced only when the damage is repaired. The length of time for repair both in hPSCs and in somatic cells was followed by determining the protein production. We have shown superior DNA repair ability and elevated protection against DNA damage in hPSCs compared to somatic cells for ultraviolet light and oxidative damage, two common sources of damage in cells. A major pathway for joining double-strand DNA breaks in mammalian cells, non-homologous end-joining (NHEJ) repair (error prone), is greater in H9 cells than in iPSCs. Another way to repair double-strand DNA breaks that uses similar (i.e., homologous) sequences is lower in iPSCs compared to hESCs and somatic cells. Further study of these repair pathways is warranted, since several methods can be used to form iPSCs. Therefore, the genomic stability for iPSCs could depend on the method used for their generation.
  • DNA repair analysis is critical to understanding how hPSCs protect against damage, but if left unrepaired, cells can turn damage into mutations when the damage is copied by enzymes (DNA polymerases) before repair occurs. Therefore, to monitor the mutations that ultimately lead to cancer or alter hPSC biology, we are using a plasmid that is damaged outside the cells and will make copies in hPSCs and somatic cells. That plasmid is introduced into cells and then the copies are recovered. The number of mutations found in the plasmid DNA indicates the likelihood of observing mutations in hPSCs compared to mutations in somatic cells. Together, these results will yield data on the stability of hPSCs and also a basis to monitor cells for stability which could serve as an indicator of safety for clinical use.

Using human embryonic stem cells to treat radiation-induced stem cell loss: Benefits vs cancer risk

Funding Type: 
SEED Grant
Grant Number: 
RS1-00413
ICOC Funds Committed: 
$625 617
Disease Focus: 
Cancer
Neurological Disorders
Skeletal Muscle
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
A variety of stem cells exist in humans throughout life and maintain their ability to divide and change into multiple cell types. Different types of adult derived stem cells occur throughout the body, and reside within specific tissues that serve as a reserve pool of cells that can replenish other cells lost due to aging, disease, trauma, chemotherapy or exposure to ionizing radiation. When conditions occur that lead to the depletion of these adult derived stem cells the recovery of normal tissue is impaired and a variety of complications result. For example, we have demonstrated that when neural stem cells are depleted after whole brain irradiation a subsequent deficit in cognition occurs, and that when muscle stem cells are depleted after leg irradiation an accelerated loss of muscle mass occurs. While an increase in stem cell numbers after depletion has been shown to lead to some functional recovery in the irradiated tissue, such recovery is usually very prolonged and generally suboptimal.Ionizing radiation is a physical agent that is effective at reducing the number of adult stem cells in nearly all tissues. Normally people are not exposed to doses of radiation that are cause for concern, however, many people are subjected to significant radiation exposures during the course of clinical radiotherapy. While radiotherapy is a front line treatment for many types of cancer, there are often unavoidable side effects associated with the irradiation of normal tissue that can be linked to the depletion of critical stem cell pools. In addition, many of these side effects pose particular threats to pediatric patients undergoing radiotherapy, since children contain more stem cells and suffer higher absolute losses of these cells after irradiation.Based on the foregoing, we will explore the potential utility and risks associated with using human embryonic stem cells (hESC) in the treatment of certain adverse effects associated with radiation-induced stem cell depletion. Our experiments will directly address whether hESCs can be used to replenish specific populations of stem cells in the brain and muscle depleted after irradiation in efforts to prevent subsequent declines in cognition and muscle mass respectively. In addition to using hESC to hasten the functional recovery of tissue after irradiation, we will also test whether implantation of such unique cells holds unforeseen risks for the development of cancer. Evidence suggests that certain types of stem cells may be prone to cancer, and since little is known regarding this issue with respect to hESC, we feel this critical issue must be addressed. Thus, we will investigate whether hESC implanted into animals develop into tumors over time. The studies proposed here comprise a first step in determining how useful hESCs will be in the treatment of humans exposed to ionizing radiation, as well as many other diseases where adult stem cell depletion might be a concern.
Statement of Benefit to California: 
Radiotherapy is a front line treatment used in California for many types of cancer, including brain, breast, prostate, bone and other cancer types presenting surgical complications. Treatment of these cancers through the use of radiation is however, often associated with side effects caused by the depletion of critical stem cell pools contained within non-cancerous normal tissue. While radiotherapy is clearly beneficial overall, many of these side effects have no viable treatment options. If we can demonstrate that human embryonic stem cells (hESC) hold promise as a safe therapeutic agent for the treatment of radiation-induced stem cell depletion, then cancer patients may have a new treatment for countering many of the debilitating side effects associated with radiotherapy. Once developed this new technology could position California to attract cancer patients throughout the United States, and the state would clearly benefit from the increased economic activity associated with a rise in patient numbers.
Progress Report: 
  • We have undertaken an extensive series of studies to delineate the radiation response of human embryonic stem cells (hESCs) and human neural stem cells (hNSCs) both in vitro and in vivo. These studies are important because radiotherapy is a frontline treatment for primary and secondary (metastatic) brain tumors. While radiotherapy is quite beneficial, it is limited by the tolerance of normal tissue to radiation injury. At clinically relevant exposures, patients often develop variable degrees of cognitive dysfunction that manifest as impaired learning and memory, and that have pronounced adverse effects on quality of life. Thus, our studies have been designed to address this serious complication of cranial irradiation.
  • We have now found that transplanted human embryonic stem cells (hESCs) can rescue radiation-induced cognitive impairment in athymic rats, providing the first evidence that such cells can ameliorate radiation-induced normal-tissue damage in the brain. Four months following head-only irradiation and hESC transplantation, the stem cells were found to have migrated toward specific regions of the brain known to support the development of new brain cells throughout life. Cells migrating toward these specialized neural regions were also found to develop into new brain cells. Cognitive analyses of these animals revealed that the rats who had received stem cells performed better in a standard test of brain function which measures the rats’ reactions to novelty. The data suggests that transplanted hESCs can rescue radiation-induced deficits in learning and memory. Additional work is underway to determine whether the rats’ improved cognitive function was due to the functional integration of transplanted stem cells or whether these cells supported and helped repair the rats’ existing brain cells.
  • The application of stem cell therapies to reduce radiation-induced normal tissue damage is still in its infancy. Our finding that transplanted hESCs can rescue radiation-induced cognitive impairment is significant in this regard, and provides evidence that similar types of approaches hold promise for ameliorating normal-tissue damage throughout other target tissues after irradiation.
  • A comprehensive series of studies was undertaken to determine if/how stem cell transplantation could ameliorate the adverse effects of cranial irradiation, both at the cellular and cognitive levels. These studies are important since radiotherapy to the head remains the only tenable option for the control of primary and metastatic brain tumors. Unfortunately, a devastating side-effect of this treatment involves cognitive decline in ~50% of those patients surviving ≥ 18 months. Pediatric patients treated for brain tumors can lose up to 3 IQ points per year, making the use of irradiation particularly problematic for this patient class. Thus, the purpose of these studies was to determine whether cranial transplantation of stem cells could afford some relief from the cognitive declines typical in patients afflicted with brain tumors, and subjected to cranial radiotherapy. Human embryonic (hESCs) and neural (hNSCs) stem cells were implanted into the brain of rats following head only irradiation. At 1 and 4 months later, rats were tested for cognitive performance using a series of specialized tests designed to determine the extent of radiation injury and the extent that transplanted cells ameliorated any radiation-induced cognitive deficits. These cognitive tasks take advantage of the innate tendency of rats to explore novelty. Successful performance of this task has been shown to rely on intact spatial memory function, a brain function known to be adversely impacted by irradiation. Our data shows that irradiation elicits significant deficits in learning and spatial task recognition 1 and 4-months following irradiation. We have now demonstrated conclusively, and for the first time, that irradiated animals receiving targeted transplantation of hESCs or hNSCs 2-days after, show significant recovery of these radiation induced cognitive decrements. In sum, our data shows the capability of 2 stem cell types (hESC and hNSC) to improve radiation-induced cognitive dysfunction at 1 and 4 months post-grafting, and demonstrates that stem cell based therapies can be used to effectively to reduce a serious complication of cranial irradiation.

Screening for Oncogenic Epigenetic Alterations in Human ES Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00408
ICOC Funds Committed: 
$685 000
Disease Focus: 
Cancer
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Embryonic stem cell-based therapies hold great promise for the treatment of many human diseases. These therapeutic strategies involve the culture and manipulation of embryonic stem cells grown outside the human body. Culture conditions outside the human body can encourage the development of changes to the cells that facilitate rapid and sustained cell growth. Some of these changes can resemble abnormal changes that occur in cancer cells. These include “epigenetic” changes, which are changes in the structure of the packaging of the DNA, as opposed to “genetic” changes, which are changes in the DNA sequence. Cancer cells frequently have abnormalities in one type of epigenetic change, called “DNA methylation”. We have found that cultured embryonic stem cells may be particularly prone to develop the type of DNA methylation abnormalities seen in cancer cells. A single rogue cell with DNA methylation abnormalities predisposing the cell to malignancy can jeopardize the life of the recipient of stem cell therapy. We have developed highly sensitive and accurate technology to detect DNA methylation abnormalities in a single cell hidden among 10,000 normal cells. In this seed grant, we propose to screen DNA methylation abnormalities at a large number of genes in different embryonic stem cells and compare their DNA methylation profiles to normal and cancer cells. This will allows us to identify the dangerous DNA methylation abnormalities most likely to occur in cultured embryonic stem cells. We will then develop highly sensitive assays to detect these DNA methylation abnormalities, using our technology. We will then use these assays to determine ES cell culture conditions and differentiation protocols most likely to cause these DNA methylation abnormalities to arise in cultured ES cells. The long-term benefits of this project include 1) an increased understanding of the epigenetics of human embryonic stem cells, 2) insight into culture conditions to avoid the occurrence of epigenetic abnormalities, and 3) a technology to monitor for epigenetic abnormalities in ES cells intended for introduction into stem cell therapy patients.
Statement of Benefit to California: 
The successful implementation of human embryonic stem cell therapy will require rigorous quality control measures to assure the safety of these therapies. Cells cultured outside the human body are known to be at risk of developing abnormalities similar to those found in cancer cells. Since a single rogue cell hidden among thousands of normal cells could cause cancer in an embryonic stem cell therapy recipient, it will be essential to have highly sensitive and accurate assays to detect these abnormalities in cultured embryonic stem cells before they are introduced into the patient. The goal of this proposal is to develop such sensitive and accurate assays. The citizens of the State of California will benefit from the availability of such assay technology to help assure the safety of human embryonic stem cell therapies.
Progress Report: 
  • Embryonic stem cell-based therapies hold great promise for the treatment of many human diseases. These therapeutic strategies involve the culture and manipulation of embryonic stem cells grown outside the human body. Culture conditions outside the human body can encourage the development of changes to the cells that facilitate rapid and sustained cell growth. Some of these changes can resemble abnormal changes that occur in cancer cells. These include epigenetic changes, which are changes in the structure of the packaging of the DNA, as opposed to genetic changes, which are changes in the DNA sequence. Cancer cells frequently have abnormalities in one type of epigenetic change, DNA methylation. In this grant, we screened for DNA methylation abnormalities at a large number of genes in different embryonic stem cells and compare their DNA methylation profiles to normal and cancer cells. This allowed us to identify potentially dangerous DNA methylation abnormalities, which occur in cultured embryonic stem cells. In the first year of this seed grant, we have developed a custom microarray to screen for DNA methylation changes at predisposed genes. In addition, we have analyzed DNA methylation in embryonic stem cells at more than 14,000 genes on a generic platform. This has allowed us to identify hundreds of genes that are abnormally methylated in various types of human cancers, and that show some evidence of this alteration in ES cells.
  • In the last phase of our study, we have screened the DNA methylation level of 1,536 genes in 142 different human embryonic stem cell pairs. Each member of the pair differed in the length of time it was in culture. Thus, our sample set was comprised of 284 paired specimens, one derived from an early passage and one derived from a late passage.
  • Our results indicate that the levels of DNA methylation varied considerably at a significant portion of the screened genes, some of which gained and some of which lost DNA methylation. These results indicate that DNA methylation in human embryonic stem cells seems to be susceptible to change over, at least in the genes examined in this study. Overall, our results suggest that the monitoring of DNA methylation changes in human embryonic stem cells may have to be incorporated as a routine protocol in stem cell manipulation.
  • During the past 12 months we have made significant progress on the data analysis of 141 paired (early passage-late passage) human embryonic stem cell lines (HESCs). The data in question was generated using a custom Illumina GoldenGate array of known Polycomb targets in HESCs, as described by Lee et al 2006. Briefly, we profiled the DNA methylation status of 1,536 loci on 282 specimens. This profiling was used to determine whether DNA methylation changes in HESCs arise as a result of time in culture at the examined loci. This determination was made by comparing the DNA methylation status of a sample of an early passage line with a late passage sample of the same line.
  • Interestingly, we found that DNA methylation in Polycomb target genes is highly affected by time in culture in a cell line-specific manner. That is, in some cell lines few DNA methylation changes were observed, while in the majority of them a large number of loci showed either an increase or decrease in DNA methylation. Via collaboration with the University of Sheffield, we were able to determine that DNA methylation instability seems to be independent of genetic instability. Furthermore, genetic instability seems to be a function of passage time in culture.

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