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Final Report Summary - MODNEURDEVDIS (Title: Self-Renewal, Fate Potential and Plasticity of Human Embryonic and Induced Pluripotent Stem Cell-Derived Neural Stem cells)

1. Challenges and objectives of the project
1.1. Short synopsis of state of the art and main objectives
Neural stem cells (NSCs) can be cultured in vitro for extensive periods of time while retaining ability to develop into different neural cell types of the nervous system. NSCs can be obtained from model animals such as mice, or from human brain tissue. In addition, NSCs can be generated from pluripotent stem cells (PSCs) – cells with ability to differentiate into all tissue types in the body. However, NSCs derived by all aforementioned techniques continuously change their properties when put in culture in the laboratory. This changing nature of NSCs does impedes their use for regenerative applications such as future cell transplantation, disease modelling, and for discovering new drugs.

1.2. Main project objective and significance
One long-term goal is to use human PSCs for developing strategies for obtaining high-purity NSC types. This will facilitate future cell replacement-based therapies, will assist in modeling human normal development and pathogenesis of neurodegenerative diseases in the Petri dish, and will enhance drug-discovery. We were specifically interested in expanding our knowledge on the dynamics of how the earliest NSCs change their features in culture. The main purpose was to constantly look for new ideas of how we can keep earliest NSCs identical and homogeneous in culture for longer periods of time, without loosing their earliest properties.

Our main strategy is to follow early NSCs day by day and week after week and record information with respect to cellular, morphological, and epigenetic (gene expression) properties of the different NSC types as they progress in culture. After collecting and analysis of such data, we can better develop new ways to prevent the earliest properties of NSCs from changing in culture – resulting in perpetuating early NSCs that hold the information to generate many nervous system cell types.

1.3. Specific objectives
Aim1 – Tracking and identifying the epigenetic changes (changes in gene expression) that NSCs undergo during culture.
Aim2 – Using this information to manipulate NSCs in culture.
Aim3 – Developing new ways to retain NSC identity in culture.

2. Our findings
2.1. Milestone 1 (for Aim1): We established a long-term neural culture of NSCs from human embryonic stem cells as a platform for tracking NSCs over time
We generated a genetic human embryonic stem cell line that lights up whenever NSCs are generated. This way we were able to systematically identify NSCs during culture and also collect them based on their “light” (green color).

2.2. Milestone 2 (for Aim1): We identified, isolated and characterized distinct types of NSCs over a period of 220 days in culture.
We next isolated growing NSCs every several days to several weeks for a period of 220 days (roughly equals to 7 months of human embryonic development). We noticed that each of the NSC types is different in many aspects: morphologically, molecularly, and importantly, also the ability to develop into different regions of the CNS. Each NSC type was able to develop to a different region in the human cortex.

We were first able to isolate early neuroepithelial cells – the earliest NSCs, which can give rise to many brain regions. We next used these early NSCs cells to obtain all other types of cells in a consecutive manner.
Most remarkably, together, all these cell types were able to reconstitute the regions that show up during entire cortical development.

2.3. Milestone 3 (for Aim1): Epigenetic characterization of NSC populations
We extensively characterized the epigenetic differences between all types of NSCs that build the cortex. We now have validated lists of genes that are activated in each of the NSC types. This is a remarkable list of the genetic pathways that NSCs undergo in order to generate the cortex.

2.4. Milestone 4 (for Aim2-3): Gain and loss of function studies to identify candidate genes governing NSC progression.
We next took these gene lists that are unique to each and every NSC type and knocked them down at the each of the different NSC types themselves. By doing so, we were able to validate which of the genes we have identified really play a critical role in governing the identity of each NSC type. This has led to a conclusive validated list of specific genes that are activated or repressed during the generation of specific NSC types that generate specific regions during the development of the cortex.

2.5. Milestone 5 (for Aim2-3): Identifying culture conditions for generation of homogeneous cortical neural stem cells.
Here, we re-analyzed all the data we obtained, in order to identify which signaling pathways are activated or shut down during emergence of the earliest NSCs of the developing cortex. After identification of which signaling pathways are either required or must not be active during generation of early NSCs, we used small molecules that can activate ort shut down these pathways. By doing so, we succeeded to manipulate pluripotent stem cells so that they

We then used small molecules We more accurately assessed the cellular and molecular heterogeneity of cortical NSCs – and to test whether we can ”program” hESCs into homogeneous early cortical progenitors by defined culture conditions. By analyzing pathway activation and repression during cortical differentiation based on our datasets, we have identified a combination of pathways that needs to be active or shut off at a specific developmental time window in vitro in order for the cortical program to commence at high efficiency and enhanced purity. Using a combination of pathway inhibition and activation, we were able to streamline and accelerate derivation of highly homogeneous cortical stem cells from hESCs. This streamlined and robust protocol marks a major step towards generating more clinically relevant purified cortical NSC populations. We confirmed this method on both mouse and human PSCs and in variety of neural differentiation schemes. Most importantly, we applied our method also on a recently developed method for generating cerebral organoids - self-assembled 3D structures that mimic fundamental cytoarchitectural and developmental aspects of corticogenesis in health and disease. The modification of organoid formation according to our new findings resulted in clearance of many of the undesired side effects of that - leading to dramatic reduction of the heterogeneity and the generation of an enhanced system for understanding corticogenesis.

2.6. Milestone 6: Modelling disease using “programmed” homogeneous cortical NSCs.
Lastly, we implemented our method to cortical progenitors and cerebral organoids from PSCs carrying an autosomal recessive Microcephaly mutation (generated by CRISPR technology in our lab). With our combined method we were able to follow abnormal corticogenesis in culture more robustly, leading to the description of novel early pathology of Microcephaly cortical NSCs.

3. The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far):
The information we have obtained from our studies (Aim 1,2) has already allowed us to develop culture conditions (Aims 2, 3) to generate the earliest cortical progenitors - neuroepithelial stem cells - and their derivatives, from hESCs. We are now extensively exploring how we can get these cells from essentially any cells type: from hESCs to hiPSCs (as we recently succeeded), or from somatic differentiated cells (by direct re-programming with defined TFs). This should bring a significant impact to the scientific community, which despite 20 years of NSC research, is still challenged by the difficulty to generate homogeneous populations of neural stem / progenitor cells. This progress should ultimately allow many laboratories that are not experienced in growth of NSCs to generate and maintain distinct, purified, homogeneous neural stem cell populations, for understanding human nervous system development, for modeling human nervous system disorders and diseases, and for screening for new drugs.


SECTION 3: Objectives for the Period

1. Overview of the project objectives for the reporting period in question
Aim1. Defining heterogeneity, stem cell and patterning potential and epigenetic state in human ES derived neural stem cell subpopulations using BAC transgenesis.
Here we will generate BAC transgenic hESC reporter lines to identify subpopulations within R-NSCs. We will further dissect the potential of R-NSC subpopulations by probing their identity, function and patterning potential following reporter-based prospective isolation. We will use live imaging for controlled observation and analysis of various neural reporters for long periods during neural induction and specification. Finally, we will also analyze chromatin state and DNA methylation maps to further identify key developmental candidate regions that may be critical for neural temporal and positional restriction.

Sub-Aims:
1A. Generating the tools - BAC transgenic hESC lines to identify subpopulations within R-NSCs.
1B. Dissecting the potential - Probing R-NSC identity, function and patterning potential in prospectively isolated R-NSC subpopulations
1C. Establishing epigenetic state for R-NSC subpopulations – towards an R-NSC signature platform for reprogramming.

Aim2. Manipulating neural stem cell state and potency in human ES cells – towards a universal neural stem cell identity.
Most of our candidate R-NSC markers are transcription factors. It is likely that some of these genes may have key functional roles in either inducing, maintaining or affecting the patterning potential of the R-NSC stage. To test this hypothesis we will perform gain of function (GOF) and loss of function (LOF) studies using lentiviral vectors targeting the candidate R-NSC genes in our BAC transgenic reporter lines established in Aim B.1.2.1.

Sub-Aims:
2A. Extrinsic Impacts - Signaling pathways
2B. Intrinsic Impacts - Gain and loss of function studies to identify candidate R-NSC and patterning genes governing NSC progression

Aim3. In vitro epigenetic reprogramming to R-NSCs by defined factors.
Here we will define the starter gene pool for optimal reprogramming by using lessons from literature and gain/loss of function. We will attempt to maintain early fates in parallel with attempt to fully reprogram across NSC stages within the neural lineage. Finally, epigenetic signature will be correlated to reprogramming efficiency.


SECTION 4: Work Progress for the Period
1. A summary of progress towards objectives and details for each task
Aim1A.
We used BAC transgenic technique as planned. We realized that despite their isolation, R-NSCs were still very heterogeneous, making the study challenging. Therefore we used a reporter for all stem cells stages– the HES5 gene. This enabled a wide range of manipulating opportunities as it resulted in molecular and cellular characterization of several NSC populations.

Aim 1B.
Using Notch activation pathway as reporter, we first generated and isolated from PSCs the earliest neuroepithelial (NE) cells of the developing nervous system known to combine high proliferative capacity and broad CNS developmental potential. We then used these cells as a starting population and followed them over more than 220 days, and using the transgenic reporter line for Notch activation, we succeeded to prospectively isolate 4 more novel types of neural stem / progenitor cells: 1) ventricular zone-like early radial glial (E-RG) cells; 2) mid neurogenesis sub-ventricular zone-like radial glial (M-RG) cells; 3) late subventricular zone radial glial (L-RG) cells; 4) adult sub ependymal zone-like Long-term neural progenitor (LNP) cells. We showed that transition through these progenitor populations is correlated with a change in their potential in culture. Remarkably, all 5 consecutively derived NSC types recapitulated the building blocked of the developing cortex: i.e. appropriate sequential generation of neurons for all cortical layers that is followed by emergence of glial cells, and culminating in the appearance of adult-like neural stem cells. Essentially, all neuronal and glial derivatives appeared in the correct order as found for cortical layers in vivo.

Aim 1C.
We succeeded in establishing epigenetic maps of all NSC populations, including DNA methylation, histone modification and RNA expression. This was done in collaboration with Harvard University. This provided a clear picture of extensive epigenetic remodeling during the progression period that is accompanied by changes in transcriptional expression pattern as well as in transcription factor binding sites in a stage specific manner. We are now able to infer or predict TF networks that drive NSC establishment and progression in vitro.

Aim 2 combined with Aim 3.
We identified 200 TFs that changed their expression during our long-term differentiation period (200 days), and which also contain all the TFs that are predicted to drive the progression of neuroepithelial cells through distinct NSC types.

In Aim 2B combined with Aim 3, we functionally validated which of the inferred TFs govern each of the NSC stages. To this end we performed an shRNA screen for gain and loss of Notch activity using lentiviral vectors targeting the 200 candidate genes. We functionally validated the genes and now have come up with the list of major TFs that drive differentiation of neural progenitors involved in CNS establishment and cortical development. While we have not yet used overexpression of combined genes to reprogram somatic cell types into distinct NSC stages, we did succeed in identifying by this shRNA screen groups of genes that are essential for Notch activation at each of the NSC stages.

In Aim 2A combined with Aim 3, we exploited the extensive transcriptional and epigenetic profiles and functional validation obtained for our highly distinct NSC / progenitor cell populations, to more accurately assess the cellular and molecular heterogeneity of cortical NSCs. We tested whether we can ”program” hESCs into homogeneous early cortical progenitors by defined culture conditions. By analyzing pathway activation and repression during cortical differentiation based on our datasets, we have identified a combination of pathways that needs to be active or shut off at a specific developmental time window in vitro in order for the cortical program to commence at high efficiency and enhanced purity. Using a combination of pathway inhibition and activation, we were able to streamline and accelerate derivation of highly homogeneous cortical stem cells from hESCs. This streamlined and robust protocol marks a major step towards generating more clinically relevant purified cortical NSC populations. We confirmed this method on both mouse and human PSCs and in variety of neural differentiation schemes. Most importantly, we applied our method also on a recently developed method for generating cerebral organoids - self-assembled 3D structures that mimic fundamental cytoarchitectural and developmental aspects of corticogenesis in health and disease. The modification of organoid formation according to our new findings resulted in clearance of many of the undesired side effects of that - leading to dramatic reduction of the heterogeneity and the generation of an enhanced system for understanding corticogenesis.

2. Impact of the research
2.1 Scientific and technical impact
This is the first time that distinct radial glial cell types are identified and isolated from human pluripotent stem cell, and are longitudinally characterized for cell fate and transcriptional identity. Therefore, the datasets generated should provide unique marker sets, which will offer crucial insights into the transcriptional networks that govern neuroepithelial cells and their transition through distinct radial glial cells. The comprehensive epigenetic data generated by this study suggest that stage specific genes may play critical roles in the generation of distinct NSC states, and thus may be useful in directly inducing or manipulating NSC states. The future use of genetic reporters specific for distinct radial glial populations offers powerful tools for harnessing the full potential of NSC biology and for applications in regenerative medicine.

We believe that in light of the exponentially growing iPSC and epigenetic reprogramming fields, this work will be extremely valuable, as it will enable generation of meaningful results when modeling development and pathogenesis of nervous system disorders, as well as for developing protocols for generating clinically relevant cells for many devastating neurological disorders such as lissencephaly, microcephaly, autism and schizophrenia.

2.2. Commercial and social impact
The ability to decipher signaling pathways that play key roles specifically in each developmental stage led us to identify 4 signaling pathways, which their manipulation accelerates the derivation of cortical stem cells from both mouse and human pluripotent stem cells. The manipulation of these pathways by growth factors and small molecules should mature into patenting (together with RAMOT - Tel Aviv University Company) this powerful tool to generate cortical stem cells from ES cells as well as patient specific induced pluripotent stem cells.

2.3. Transfer of knowledge to the host institute
The knowledge that was gathered in the passing 4 years has generated several avenues of transfer of knowledge to the host institute. First, we published three papers affiliated to Tel Aviv University and have one additional manuscript under review. Second, we presented our work orally 4 times and as poster 3 times in major stem cell scientific meetings. We help other laboratories on a regular basis, particularly in growing and differentiating pluripotent stem cells into the neural lineages. Finally, as a lecturer in the Faculty I established an Introduction to Stem cell Biology course for the Graduate Program in the Faculty. This course is usually packed and I feel extremely satisfied to transfer the latest literature update on stem cell topics including published and unpublished data from our lab.

2.4. Ethical issues
2.4.1. Applying the 3R Principle in animal research
Although we have provided detailed information on the numbers and nature of the experiments with mice, we eventually used mainly tissue culture procedures from cell lines for the entire research period. In fact, we only used 20 timed-pregnant mice at the period of 2010-2012 (See attached legal authority documentation on use of animals) and have not renewed our animal approval application since 2012 (see same document). By this we have strongly applied the first 3R Principle - Replacement – which urges for replacing animal use with other non-animal use procedures, when possible.

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