Pluripotent stem cell-based paradigms to study brain stem cell dynamics in development, disease and tumorigenesis

Dr. Yechiel Elkabetz

October 13, 2021

Our focus areas are human cortical development in health and disease: Neural stem cell patterning, heterogeneity and potential in development and microcephaly. Human neural stem cell ontogeny: Programming and reprogramming of neural stem cell fates. Fate determinants in pluripotency and neural lineage commitment: Lineage tracing of dividing neural stem cells. Origin of brain tumorigenesis: Molecular and cellular landmarks of medulloblastoma.
 

Research Concepts

Cultured Neural stem cells (NSCs) are in vitro building blocks for brain development that exhibit high proliferative capacity and broad developmental potential – and as such, they serve as an unlimited source for creating model systems to understand development and disease. A number of facts challenge the use of cultured NSCs for therapeutic purposes, among them NSC heterogeneity and progressive restriction in NSC developmental potential.

One of the major goals of my lab is to develop strategies for generating homogeneous sources of NSC types. Such established populations can serve as universal cellular platforms for streamlining the generation of distinct neuronal and glial populations, modelling specific brain functions in health and degeneration, standardizing pharmacological or genetic screens towards discovering novel drugs, and making pure cells for future cell replacement-based therapy. We are mainly motivated by the idea that only clinically relevant cells with accurate cell identity can produce meaningful phenotypes and tangible therapeutic solutions.

Our main strategy is inspired by the notion that distinct cell types in culture can be distinguished apart by a myriad of features that can be identified by different tools. Based on this, we devise paradigms for separation of cell types in a variety of neural differentiation strategies; we use genetic tools such as CRIPSPR genome editing, reporter gene expression and lentiviral-based gene introduction, in order get access to, isolate and manipulate unique cell types. We use CRE-lox/brainbow multiple fluorescent tagging, barcode labeling and single cell sequencing methods to label, detect, track and analyze origin of brain structures and substructures in health and disease models.

 

Scientific highlights

  • Consecutive isolation of long-term cultured NSCs from PSCs reveals distinct developmental stages that recapitulate human corticogenesis. Edri*, Yaffe* et al., Nature Communications 2015.
  • Epigenetic footprinting and functional analyses of PSC-derived cortical NSC stages reveals transcriptional networks driving cortical NSC ontogeny.Ziller*, Edri* et al., Nature 2015.
  • Quantitative live imaging of neural rosettes reveals structure-function dynamics of NSCs within rosettes coupled to corticogenesis. Ziv*, Zaritski*, Yaffe* et al., PLoS Computational Biology 2015.

 

Recently Accomplished Projects

Robust cortical neural stem cell homogeneity in cerebral organoids facilitates emergence of outer radial glial cells. Rosebrock el al., In Revision

Comparative analysis of brain cultures derived by diverse methods in health and microcephaly. Organoids (right/pink) and monolayer cultures (left/green) derived by inhibition-free, dual SMAD inhibition and combined dual SMAD/WNT inhibition. Notch activation and radial organization are both enhanced and overlapping only under combined inhibition, which yields cortical fates.

In recent years there has been an astounding awakening of brain organoid models for studying brain development and disease. This evoked a blast of highly diverse methods to derive these structures. However, these methods lack specific readouts that instruct landmarks for successful differentiation, thus challenging the validity of disease phenotypes. This reinvigorated us to revisit the basic derivation paradigms side-by-side at the transcriptional, cellular, and cytoarchitectural level. We found that a short inhibition of three signaling pathways during early organoid establishment is sufficient for yielding robust and lasting cortical identity with efficient suppression of non-cortical fates in organoid neural stem cells (NSCs). In contrast, other widely used methods are inconsistent in their cortical specification capacity, yielding NSCs with mixed regional identity. We importantly found that our method also increases outer radial glia (oRG) cells – a population uniquely responsible for brain expansion in humans. Finally, we revealed that only this method enables exposing cortical defects in a cortical disease organoid model (microcephaly). Thus, combined inhibition is critical for establishing a rich cortical cell repertoire, for enabling fundamental cytoarchitectural features of cortical development, and for meaningful disease modeling. We now use this method as a platform to better understand cortical development and disease, to identify new factors affecting NSC self-renewal and research on tumorigenicity brain cancer stem cells.

 

Ongoing and Future Projects

Human neural stem cell ontogeny: Programming and reprogramming of neural stem cell fates.

Reprogramming NSCs. An example for reverting later progressing neural progenitors into early type neural progenitors by defined factors. Under “normal” non-reprograming conditions, progenitors progress through distinct stages in vitro. Reprogramming is performed by educated guess as well as screens for molecules, TFs, shRNAs and miRs.

Cellular reprogramming towards pluripotency, directed reprogramming within and across lineages, and directed transition between primed and naïve pluripotent stem cell states - have all reinvigorated the idea of developing novel strategies for inducing and / or maintaining distinct types of homogeneous, self-renewing NSCs. Such self-perpetuating NSC types would act as “pluripotent” equivalents of the nervous system and therefore are expected to lead an exponential growth in the use of NSCs for various regenerative medicine applications.

Taking advantage of the transcriptional, epigenetic and functional characteristics of our isolated NSC types, we are building a platform to evaluate the regenerative potential of specific NSC populations and gain insights into their self-renewing mechanisms. We are generating stage specific PSC reporter lines with which we are assessing developmental potential across differentiation stages combining live cell imaging, cell sorting, transcriptomic and epigenomic approaches, and in vitro stem cell assays. Finally, we use the information gained, together with these reporters, as powerful readouts for reprogramming somatic and diverse neural stem cell populations towards specific NSC stages.

Fate determinants in pluripotency and neural lineage commitment: Lineage tracing of neural stem cells.

The multidisciplinary approaches taken in our lab provide evidence on how molecular identity, cell fate potential and cytoarchitectural dynamics of distinct NSCs are correlated with appropriate generation of the building blocks that construct the cortex. In addition to transcriptional and epigenetic mechanisms affecting cell fates, we are also interested in elucidating cytoarchitectural and cell biological mechanisms underlying cell fate decisions. We will build transgenic CRE-lox PSC reporter lines that will allow unbiased or targeted progenitor cell labeling using inducible or random recombination-based fluorescent labeling driven by a ubiquitous or specific promoters, respectively. Such a system should enable genetic marking, monitoring, isolating and characterizing unique progenitor subpopulations. Fluorescent labeling will be combined with unbiased single cell molecular barcoding. Populations will be analyzed by single cell approaches of cultured monolayers or organoids.

Origin of brain tumorigenesis: Molecular and cellular landmarks of medulloblastoma.

Brain tumors are the most common cause of childhood cancer-related deaths, and medulloblastoma is the most common malignant pediatric brain tumor. Current medulloblastoma therapy improves the overall survival, but many patients still die from their disease. Medulloblastoma is classified based on histologic and molecular features, with some subgroups exhibiting vastly different transcriptional and genomic profiles, implying that they are driven by distinctive underlying biology. The goal of this study is to generate an in vitro model which mimics the development and progression of genetic, transcriptional and cellular events observed in this human pediatric disease – towards understanding in better depth the process of tumor origin. We are generating human induced pluripotent stem cell (hiPSC) lines that carry inducible expression of duplications/mutations in known deriver genes, alongside inducible labeling of key genes suspected to act as cancer stem cells. By overexpressing driver genes and tracking suspected populations with reporter based sorting and single cell approaches we aim to reveal the mechanism behind the malignant transformation in human disease.

For more information have a look at the website of the Elkabetz lab

 

Go to Editor View