Recreating the nuclear reprogramming of embryonic stem cells in vitro.

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01292
ICOC Funds Committed: 
$0
Stem Cell Use: 
iPS Cell
Public Abstract: 
The reprogramming of skin fibroblasts into pluripotent stem (iPS) cells represents a significant milestone towards the goal of developing stem cell therapies tailored to the patient. However, the reprogramming is slow and inefficient. A key step is the ability of four exogenously introduced transcription factors to turn on or activate the endogenous versions of their own genes, a process termed autoregulation. In fibroblasts, the genes for the endogenous transcription factor genes are turned off because they are assembled into a repressive structure termed silent chromatin. The repressive structure must be removed to activate the endogenous stem cell transcription factor genes. The efficiency by which the endogenous genes are activated is poorly understood. Dozens of different proteins assist the four transcription factors during the reprogramming process. However, little is known of how the dozens of proteins coordinate their actions to remove the silent chromatin structures and replace them with active chromatin. Our goal is to study this process and use the resulting knowledge to improve the efficiency of iPS cell formation. This process can be studied by recreating it in a test tube or in vitro. This biochemical recreation involves three steps: 1. Generating highly pure versions of the four transcription factors. 2. Reproducing the silent chromatin environment of the gene. 3. And finally, identifying each of the steps involved in reactivating the endogenous genes. We have developed a technology that permits us to biochemically recreate silent chromatin on the transcription factor genes in vitro. We then attach the silent chromatin to magnetic beads, add protein mixtures from disrupted stem cells, and use magnets to capture beads with the attached proteins, which are in the process of converting silent chromatin to active chromatin. This “immobilized template capture” technique also allows us to delineate the steps involved in the reprogramming process. The proteins are identified by a state-of-the-art method known as multidimensional protein identification technology (MuDPIT). By combining the immobilized template and MuDPIT techniques, we will be able to provide detailed knowledge about how autoregulation is achieved. As such, we will be able to determine which specific steps are limiting in the conversion of fibroblast to iPS cells. Knowledge of the mechanism of stem cell gene regulation is essential for fully understanding stem cell self-renewal and the transition of fibroblasts to iPS cells. The information can be utilized to improve the efficiency of iPS cell formation.
Statement of Benefit to California: 
California is investing 6 billion dollars in stem cell research with much of the principal being spent in the first 10-15 years on research and development. To date, much research has focused on the therapeutic potential of stem cells and understanding a few fundamental aspects of how stem cells self renew and differentiate. A major gap in our understanding lies in the most basic aspect of stem cell biology, the mechanics by which stem cell genes are regulated. To fully understand and to optimally manipulate the technology, a significant amount of basic knowledge must be obtained. The lack of basic mechanistic knowledge is what initially limited gene therapy and immunotherapy from achieving clinical application. For example, in gene therapy, we knew how to design therapeutic genes but knew little about how to deliver and regulate them. The situation has improved considerably in recent years, driven not by new technologies per se, but due to an investment in understanding basic mechanisms. We are now starting the same process with stem cells. Our technology and knowledge base have improved considerably but there are large gaps. Few scientists doubt the potential. However, many overestimate our knowledge base and ability to achieve therapeutic goals. It is important that we determine at the most basic level how stem cell genes are regulated. We know that four gene regulatory factors, when added to skin fibroblasts, can convert these into stem cells. However, we do not know precisely how they do this. To date, CIRM has invested heavily in labs trying to understand the functions of these four transcription factors in living cells. However, in the gene regulation field of biology, many of the major advances derived from biochemical or in vitro studies. My laboratory works on the fundamental aspects of how genes are regulated. We have recreated key events in a test tube. We have developed new biochemical approaches for determining how stem cell genes are regulated and for understanding how the four transcription factors function when bound to chromatin. Our studies will provide insight into and understanding of the processes by which stem cells self renew and differentiate. As such, the basic knowledge will help further the transition of into stem cells into clinical applications.
Progress Report: 
  • The use of stem cells as a therapeutic tool is predicted to revolutionize many medical fields, such as tissue replacement for trauma-associated damage and aging-related diseases, and the advent of induced pluripotent stem (iPS) cells that are derived from somatic cells has generated high hopes for patient-matched cellular therapy. However, the major hurdle to the routine use of iPS cells for diagnostic or therapeutic applications is the inefficiency with which they are generated. This is largely because iPS are produced asynchronously, relatively slowly and at low frequency. An understanding of the mechanisms of nuclear reprogramming of somatic human fibroblasts to pluripotent cells that could lead to enhance the rate and frequency of reprogramming is of great fundamental and translational interest.
  • Our approach relies on our extensive experience over the past two decades using cell fusion (heterokaryons) to understand the principles inherent in the conversion of one cell fate to another. There is no cell division or nuclear fusion in these heterokaryons, ensuring that there is no loss of genetic material, and reprogramming takes place in the presence of the complete proteome. Specifically, we have applied this powerful process to study nuclear reprogramming of somatic cells toward stemness and identify a key player in the reprogramming toward stemness. Key to this approach are species differences between the fused cells that enable the gene products of the ‘reprogrammer’ (the inducer) and ‘reprogrammed’ (the responder) nuclei to be distinguished. Specifically, we have made interspecies heterokaryons between mouse ES cells and human fibroblasts in order to investigate the conversion of the somatic human cell into a pluripotent human stem cell. We analyzed the gene patterns of the singly isolated human-mouse fused cells by RT-PCR using specie-specific primers, and observed that more than 70% of the human nuclei expressed the Oct4 and Nanog genes. Furthermore, the reprogramming process is fast, as detected 24 hours after fusion. In parallel, we focused on the epigenetic modifications induced after fusion in the heterokaryons, in particular on the DNA methylation status of the promoters for the stemness genes Oct4 and Nanog. There is ample evidence that actively transcribed genes exhibit very low levels of methylation on CpG motifs while repressed genes display higher levels of methylation. Interestingly, we observed that both promoters, Oct4 and Nanog were demethylated in the human nucleus, as early as 24 hours after fusion. Next, we sought to elucidate the potential role of a key enzyme that has been recently implicated in DNA demethylation in Zebrafish. We performed in depth analysis of the role of Activation-Induced Cytidine Deaminase (AID) by loss and gain of function approaches. First, we analyzed the expression levels of AID in the human fibroblasts and in the mouse ES cells and detected significant amounts of AID in both cell types supporting our assumption that AID is important for reprogramming. Next, we designed a set of siRNAs to directly examine the function of AID in the initial steps of reprogramming in the heterokaryons, and demonstrated that knock-down of AID correlated with the inhibition of Nanog and Oct4 expression. Furthermore, we monitored the DNA methylation status of their respective promoters, and found that the inhibition of AID protein is coincident to a decrease in DNA demethylation of Oct4 and Nanog promoters. Finally, in order to show that AID per se is implicated in the inhibition of the pluripotency genes, we re-introduced the AID protein in siRNA-mediated knocked down cells, and showed that Oct4 and Nanog levels were increased and the DNA methylation is reversed.
  • In conclusion, during the first year of funding, our results demonstrated that reprogramming toward pluripotency in heterokaryons is fast and efficient and involves active DNA demethylation since there is no cell division or DNA replication. In addition, we showed that the AID enzyme, known for its role in generating antibody diversity in B cells, is a key component for reprogramming toward stemness. We are now exploring the ability of AID to speed up iPS generation. In addition, we are utilizing the heterokaryon system to identify and test other early regulators by studying the gene expression changes at a global level.
  • Induced pluripotent stem cells (iPS) can be produced from virtually any somatic cell by the overexpression of a few transcription factors, a process termed “nuclear reprogramming”. However, the generation of iPS is slow (2 weeks) and the frequency of somatic cells which undergo successful reprogramming is very low (0.1-1%). At present, the molecular mechanisms underlying reprogramming are not well understood. This is in large part due to an inability to analyze early stages of reprogramming at the molecular level in populations which are heterogeneous or where cell numbers are limiting. We hypothesized that the inefficiency of reprogramming to iPS is due to as yet unidentified molecular regulators or pathways critical to the early onset of reprogramming.
  • In order to study the molecular mechanisms of reprogramming, a different experimental system was needed; one with a highly efficient, rapid onset of reprogramming. Our previous research (Bhutani et al, Nature 2010) showed the development of a synchronous, high efficiency, rapid reprogramming approach consisting of heterokaryons (interspecies multinucleate fused cells). In these multinucleate cells, activation of human pluripotency genes such as Oct4 and Nanog occurs rapidly (24hrs) and efficiently (70% of single heterokaryons). During the first year of funding, our results demonstrated that reprogramming toward pluripotency in heterokaryons is fast and efficient and involves active DNA demethylation since there is no cell division or DNA replication. In addition, we showed that the AID enzyme, known for its role in generating antibody diversity in B cells, is a key component for reprogramming human somatic cells towards pluripotency.
  • Now during our second year of funding, we are testing for both the requirement of AID for iPS generation but also the ability of AID to speed up iPS generation. We also reasoned that global RNAsequencing of heterokaryons would provide us with further insight into the early reprogramming process and are utilizing the heterokaryon system to identify and test other early regulators by studying gene expression changes genome wide. We now have optimized methodologies which allow us to accomplish this aim and have performed global RNA-seq at 6hr, day 1, day 2, and day 3 post-heterokaryon formation. We are now beginning to analyze for early activated genes either related to pluripotency network associated transcription factors or epigenetic modifiers. More specifically, we are interested in enzymes that are involved in DNA demethylation and are in the concluding process of validating AID in iPS generation.
  • The speed and efficiency of reprogramming in the heterokaryon system provides a means to identify critical transcription factors and cellular pathways involved in early reprogramming. Our research with heterokaryons enables mechanistic insights into the process of nuclear reprograming which are not possible to identify using iPS.
  • Induced pluripotent stem cells (iPS) can be produced from virtually any somatic cell by the overexpression of a few transcription factors, a process termed “nuclear reprogramming”. However, the generation of iPS is slow (2 weeks) and the frequency of somatic cells which undergo successful reprogramming is very low (0.1-1%). At present, the molecular mechanisms underlying reprogramming are not well understood. This is in large part due to an inability to analyze early stages of reprogramming at the molecular level in populations which are heterogeneous or where cell numbers are limiting. We hypothesized that the inefficiency of reprogramming to iPS is due to as yet unidentified molecular regulators or pathways critical to the early onset of reprogramming.
  • In order to study the molecular mechanisms of reprogramming, a different experimental system was needed; one with a highly efficient, rapid onset of reprogramming. Our previous research (Bhutani et al, Nature 2010) showed the development of a synchronous, high efficiency, rapid reprogramming approach consisting of heterokaryons (interspecies multinucleate fused cells). In these multinucleate cells, activation of human pluripotency genes such as Oct4 and Nanog occurs rapidly (24hrs) and efficiently (70% of single heterokaryons). During the first year of funding, our results demonstrated that reprogramming toward pluripotency in heterokaryons is fast and efficient and involves active DNA demethylation since there is no cell division or DNA replication. In addition, we showed that the AID enzyme, known for its role in generating antibody diversity in B cells, is a key component for reprogramming human somatic cells towards pluripotency.
  • Now during our third year of funding, we have both demonstrated the requirement of AID for iPS generation but also the ability of AID to increase iPS generation by roughly two fold. Moreover, because we had reasoned that global RNA-sequencing of heterokaryons would provide us with further insight into the early reprogramming process, we now have optimized methodologies which allow us to accomplish this aim and have performed global RNA-seq at 6hr, day 1, day 2, and day 3 post-heterokaryon formation. Through this analysis we have now identified a secreted factor identified via RNA sequencing in Heterokaryons that can substitute for one of the key iPS reprogramming factors, c-myc. The substitution of myc by a secreted factor allows for the generation of safer patient derived iPS cells by relieving the need for viral integration of the potent oncogene c-myc.
  • In sum, the speed and efficiency of reprogramming in the heterokaryon system provides a means to identify critical transcription factors and cellular pathways involved in early reprogramming. Our research with heterokaryons enables mechanistic insights into the process of nuclear reprograming which are not possible to identify using iPS.

© 2013 California Institute for Regenerative Medicine