Molecular determinants of neural stem cell differentiation

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01292
ICOC Funds Committed: 
$0
Stem Cell Use: 
iPS Cell
Public Abstract: 
Neurodegenerative diseases comprise a heterogeneous spectrum of neural disorders that cause severe and progressive cognitive and motor deficits. A histological hallmark of these disorders is the occurrence of disease-specific cell death in specific regional subpopulations of neurons, such as the loss of cholinergic neurons in Alzheimer’s disease, gaba-ergic interneurons in various forms of Batten’s disease, spiny interneurons of the basal ganglia in some forms of metabolic disease, etc. Neurodegenerative disease can also possibly occur from the loss or dysfunction of selected glial cell subsets, such as the dysfunction of supportive glial cells around somatic motor neurons in amyotrophic lateral sclerosis. Differentiation of human pluripotent stem cells into cells of the neural lineage, therefore, has become a central focus of a number of laboratories. This has resulted in the description in the literature of several dozen methods for neural cell differentiation from human pluripotent stem cells. Among the problems associated with this are the wide variability of neural differentiation potential of different PSC lines and the lack of comparison of the resulting neural cells to those derived from the brain itself. PSCs, because of their broad neuro-developmental potential, are expected to help provide a therapeutic cure for a wide variety of neurodegenerative diseases. To achieve this expectation, we need to identify all the factors involved in neural differentiation such that our understanding of the mechanisms involved becomes more complete. Moreover, we need to be capable of manipulating differentiation pathways such that desired subtypes of neuronal progenitors can be selected that will provide functional phenotypes upon transplantation. We are taking a whole genome expression analysis approach to help identify markers and networks of gene associated with distinct stages of neural differentiation and also inhibition of neural differentiation. We hypothesize that once such factors are identified, we may be able to more readily generate and isolate a transplantable population of neural cells.
Statement of Benefit to California: 
Current conservative estimates indicate that at least 16 million individuals in the US (2 million in California alone) are afflicted and currently living with a brain disease. This incidence may be higher as the estimates exclude rare disorders and childhood neurological disorders such as neuro-metabolic diseases and autism. An estimated 16% of California households may be dealing with the care of a loved one with brain disease. Many of the diseases (Alzheimer’s, Stroke and Parkinson’s) that affect the brain are progressive and their incidence and prevalence increase with age. By 2020, it is estimated that almost 1 million people will be aged 85+ in California alone, with a high proportion (36%) having moderate or severe neurological function. This represents an immense challenge to California’s health care system. Neural stem cell (NSC) populations have great potential for revolutionizing medicine by providing successful neuroprotective or regenerative therapy for brain disease following transplantation. The use of neural stem cells in the clinical therapy of brain disease and injury continues to remain an area of intense focus. The recent groundbreaking work of the derivation of induced pluripotent stem cells (iPSCs) from human somatic cells has additionally created the reality of deriving immune matched NSCs from adult cells such as skin. However, difficulties in the development of these potential therapies relate to insufficient tools to isolate, identify and characterize NSC populations. We propose to further develop existing molecular pathway analysis tools to identify a "NeuroNet" or molecular fingerprint (s) specific for NSC and neural induction/differentiation pathways from embryonic - derived NSCs, brain - derived NSCs and iPSC - derived NSCs. Realizing the full potential of all such NSCs as a source of defined cells for cell based neurological therapies will ultimately require a critical in - depth knowledge of factors present within these cells that are responsible for inducing an early neural phenotype and for orchestrating differentiation down specific neural lineage(s). Defining the NeuroNet will be instrumental in facilitating both new discoveries in neural development and providing a means of simplifying characterization and quality control of these cells and, most importantly, guiding neural differentiation into clinically useful cell types.
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