Muscular Dystrophy

Coding Dimension ID: 
302
Coding Dimension path name: 
Muscular Dystrophy

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.

Functional Genomic Analysis of Chemically Defined Human Embryonic Stem Cell

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00100
ICOC Funds Committed: 
$2 628 635
Disease Focus: 
Genetic Disorder
Muscular Dystrophy
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Regenerative medicine holds the promise that tissues can be engineered in vitro and then transplanted into patients to treat debilitating diseases. Human Embryonic Stem Cells differentiate into a wide array of adult tissue types and are thought to be the best hope for future regenerative therapies. This grant has three main goals: 1. The creation of new human embryonic stem cells in animal free conditions which will allow for future therapeutic uses. 2. The creation of human embryonic stem cell that contain mutations in their genomes that cause diseases, including cystic fibrosis, muscular dystrophy, Downs Syndrome and many others. These lines can be used to study these diseases and to test potential therapies 3. A close biological assessment of one of the first tissues to arise during differentiation of human embryonic stem cells – the endoderm. Since the endoderm eventually, during many days of development, becomes the pancreas, liver, and gut. It is critically important that we know everything about this very specialized tissue if we are to attempt to engineer these organs in the laboratory. Our overwhelming goal is to provide tools that clinician can use to treat disease whether it is to establish new and improved human embryonic stem cell lines or to find new ways of creating endodermal tissues within the laboratory for future therapeutic uses.
Statement of Benefit to California: 
This grant will provide to the state of California new human embryonic stem cell lines that could be used in future therapeutic uses. It will also provide disease specific human embryonic stem cell lines that can be used to study disease and as models to test pharmacological compounds to treat disease. We will also provide a characterization of tissues generated from the new human embryonic stem cells which we hope will someday aid in the formation of liver, lung and pancreas.
Progress Report: 
  • We have made significant progress during the previous granting period which has resulted in a publication in Genome Research detailing genomic DNA methylation changes in a variety of human embryonic stem cells and their derivatives. We have also been successful in identifying regions of the genome bound by of histone modifications and transcription factors in hESCs and derived endoderm. These targets have led to a greater understanding of how Nodal signaling and chromatin configuration maintain pluripotent state and subsequently trigger differentation. We expect 3 publications to result from this work during the next granting period.
  • Toward a goal of developing endodermal lineages from hESCs, including liver, pancreas, lung and intestine, we have developed new tools and approaches to identify these subtypes as well as a molecular understanding of how these subtypes emerge. These advances are highlighted in three papers which are currently under review for publication and one in preparation. Two of these papers redefine endodermal subtypes derived from hESCs, including new methods to isolate lineage restricted endodermal populations and a means to distinguish between single endodermal cells. The third paper provides an unprecedented view of the Nodal signaling pathway and its intersection with bivalent domains in both hESCs and derived endoderm. This chromatin signature which consists of Smad transcription factors and both histone repressive and active marks is the most conducive for mediating downstream targets of Nodal, providing inroads into how signaling pathways and chromatin cooperate during fate specification in hESCs. This analysis has led to the elucidation of new proteins that mediate Nodal signaling; ones that play a key role in endoderm specification.
  • During the past year, we have completed three research projects as a result of this grant. Two of these have recently been published in Genes and Development and Developmental Biology. The third is currently in review at Development. The discoveries reported in these papers are highlighted below:
  • HEB and E2A function as SMAD/FOXH1 cofactors
  • Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We have identified a new motif, termed SMAD Complex Associated (SCA) that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two bHLH proteins - HEB and E2A - bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Further, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation.
  • Chromatin and Transcriptional Signatures for Nodal Signaling During Endoderm Formation in hESCs
  • The first stages of embryonic differentiation are driven by signaling pathways hardwired to induce particular fates. Endoderm commitment is controlled by the TGF-β superfamily member, Nodal, which utilizes the transcription factors, SMAD2/3, SMAD4 and FOXH1, to drive target gene expression. While the role of Nodal is well defined within the context of endoderm commitment, mechanistically it is unknown how this signal is manifested at binding regions within the genome and how this signal interacts with chromatin state to trigger downstream responses. To elucidate the Nodal transcriptional network that governs endoderm formation, we used ChIP-seq to identify genomic targets for SMAD2/3, SMAD3, SMAD4, FOXH1 and the active and repressive chromatin marks, H3K4me3 and H3K27me3, in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that while SMAD2/3, SMAD4 and FOXH1 binding is highly dynamic, there is an optimal signature for driving endoderm commitment. Initially, this signature is marked by both H3K4me3 and H3K27me3 as a very broad bivalent domain in hESCs. Within the first 24 hours, at a few select promoters, SMAD2/3 accumulation coincides with H3K27me3 reduction so that these loci become relatively monovalent marked by H3K4me3. JMJD3, a histone demethylase, is simultaneously recruited to the promoters of the endodermal gene GSC and EOMES, suggesting a conservation of mechanism at multiple promoters genome-wide. The correlation between SMAD2/3 binding, monovalent formation and transcriptional activation suggests a mechanism by which SMAD proteins coordinate with chromatin at critical promoters to drive endoderm specification.
  • Distinguishing Cells with Housekeeping Transcripts
  • Distinguishing between cell types is central to a broad array of biological fields and is at the heart of advancing regenerative medicine and cancer diagnostics. In this report, we use single cell gene expression to identify transcriptional patterns emerging during the differentiation of human embryonic stem cells (hESCs) into the endodermal lineage. Endoderm specific transcripts are highly variable between individual CXCR4+ endodermal cells, suggesting that either the cells generated from in vitro differentiation are distinct or that these embryonic cells tolerate a high degree of transcript variability. Housekeeping transcripts, on the other hand, are far more consistently expressed within the same cellular population. However, when we compare the levels of housekeeping transcripts between hESCs and derived endoderm, patterns emerge that can be used to clearly separate the two embryonic cell types. We further compared 4 additional human cell types, including 293T, iPSC, HepG2 and endoderm derived iPSC. In each case the relative levels of housekeeping transcripts defined a particular cell fate. Interestingly, we find that three transcripts, LDHA, NONO and ACTB, contribute the most to this diversity and together serve to segregate all 6 cell types. Overall, this suggests that levels of housekeeping transcripts, which are expressed within all cells, can be leveraged to distinguish between human cell types and thus may serve as important biomarkers for stem cell biology and other disciplines.

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