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Flora Vaccarino, MD

Harris Professor in the Child Study Center; Director, Program in Neurodevelopment and Regeneration, Child Study Center; Professor in the Department of Neuroscience

Research Summary

Stem cells are pluripotent cells that give rise to all tissues of the body, including neurons and glia of the central nervous system. Genome-scale complex regulatory processes unfold over time and space in the prenatal central nervous system to establish developmental programs that govern the differentiation of countless neuronal and glial cell types that populate the brain. Cell-intrinsic, species-specific genetic blueprints as well as cell-to-cell contacts and signaling systems govern these complex regulatory processes and render them more robust to perturbations. We hypothesize that slight deviations from typical programs of differentiation and patterning of neural stem cells predispose to disorders such as autism, Tourette syndrome, and schizophrenia. Induced pluripotent cells (iPSC) derived from living individuals and differentiated into telencephalic organoids recapitulate forebrain development in a dish, allowing us to examine typical and atypical development in patients with neuropsychiatric disorders and the underlying gene regulatory processes. Specifically we study the impact of noncoding RNAs, histone modifications, enhancer activity, and somatic mutations on typical and atypical brain development in human brain organoid culture and, whenever possible, in vivo.

Another interest of our lab is the study of cell lineages in human development. Tracing cell lineages is fundamental for understanding the rules governing development in multicellular organisms and delineating complex biological processes involving the differentiation of cell types with distinct lineage hierarchies. For ethical reasons lineage mapping in humans must be based on retrospective methods relying on naturally occurring mosaic mutations.

Mosaic mutations occur throughout the genome in all cells starting from the first cell divisions of the embryo, are inherited by all their daughters and can be discovered by comparing the genomes of individual cells and tissues within an individual.

Specialized Terms: Neural stem cells; Cerebral cortex; Neuronal progenitors; Postmortem human brain; Induced pluripotent stem cells; somatic mosaicism, human cell lineage reconstruction, enhancer activity, gene expression, telencephalic organoids.

Extensive Research Description

The Vaccarino laboratory has elucidated crucial mechanisms that regulate neural stem cell self-renewal, survival and differentiation. The lab has been examining conserved mechanisms of forebrain development using mouse models, and human-specific mechanisms by examining human stem and progenitor cells. Using induced pluripotent cells (iPSC) derived from living individuals, the Vaccarino group is now examining how human stem cell differentiation varies across different genetic backgrounds, sex, and clinical phenotypes.

In the 1990’s Vaccarino and colleagues reported that an extracellular protein called Basic Fibroblast Growth Factor 2 (FGF2) increases the number of progenitors for excitatory cortical neurons in vitro (Vaccarino et al., 1995) and in vivo, by microinjection of FGF2 into the cerebral ventricles of rat embryos, producing an increase in cortical surface area (Vaccarino et al., 1999). This was the first evidence that a single factor can elicit a permanent increase in pyramidal neuron number and cerebral cortical size in a mammalian species. Subsequent work demonstrated that the FGF ligands (Vaccarino et al., 1999; Raballo et al., 2000; Korada et al., 2002) and FGF receptors are essential for normal telencephalic development in a region-specific fashion. The Fgfr1 gene knockout alone thwarts cell proliferation within the hippocampal primordium, causing lifelong hippocampal atrophy (Ohkubo et al., 2004), and the double knockout of Fgfr1 and Fgfr2 causes prefrontal cortex volume loss with fewer pyramidal cells (Stevens et al, 2010). Finally, the combined knockout of Fgfr1, Fgfr2 and Fgfr3 in early neurogenesis depletes the cortical stem cell pool globally, resulting in premature ending of neurogenesis and decreased cortical surface area (Rash et al, 2011). Conversely, a microinjection of FGF2 in the lateral ventricles at pre-neurogenic stages of cortical development generates a massive enlargement of frontal cortical surface and the appearance of gyrus-like convolutions in stereotypic bilateral locations (Rash et al, 2013).

In summary, our studies have shown that FGFs establish the primary structure and the surface area of the cerebral cortex by promoting the self-renewal of neural stem cells and the differentiation of projection neurons from neuroepithelial precursors.

The lab has also examined the roles of FGFs in astroglial cells—diverse cellular elements that evolve from a primary role of neural progenitors during embryonic development, to essential partners in neuronal migration, maturation, and metabolic support in the adult brain. To study astroglial cells, the lab has generated the GFAP-CreERT2 transgenic line, in which the Cre recombinase can be transiently induced by a tamoxifen injection in GFAP+ astroglial cells (Ganat et al, 2006). By marking GFAP+ astroglial cells via recombination of genetic reporters, we found that astrocytes can produce neurons in the immature brain, a process that is enhanced by hypoxic injury (Fagel et al., 2006, and Fagel et al, 2009; Bi et al, 2011) and dependent in part upon FGF receptor function (Fagel et al, 2009; Stevens et al, 2102). Furthermore, FGF receptors are required in astroglia to indirectly promote the maturation of cortical inhibitory interneurons during early postnatal development (Müller Smith et al, 2014). Induced loss of FGF signaling only in postnatal astrocytes generates stereotypic locomotor hyperactivity and learning and memory defects that correlate with deficit in specific cell populations in the postnatal brain (Müller Smith et al, 2008, Stevens et al, 2012).

Conscious of the profound differences between brain development in rodents and primates, the Vaccarino lab has been pursuing parallel studies of the human brain. A major theme is whether an excitatory/inhibitory neuron imbalance in specific forebrain systems due to disparate etiologies (i.e., gene mutations; prenatal factors; environmental noxae) may predispose to neuropsychiatric disorders such as autism and Tourette syndrome.

Through postmortem studies of patient and control brains, the lab has discovered in landmark studies that individuals with Tourette’s syndrome (TS) have losses of GABAergic and cholinergic interneurons in the striatum. TS is a developmental disorder of childhood characterized by motor and vocal tics. We demonstrated a large decrease in three classes of interneurons in the striatum of TS: Parvalbumin+; NOS+/NPY+/SST+; and cholinergic (Kalanithi et al, 2005; Kataoka et al, 2010). RNA sequencing confirmed the decreased in interneuron transcripts, and also revealed an up-regulation of inflammatory response- and immune system-related genes (Lennington et al., 2014). The hypothesis is that altered development of subsets of inhibitory neurons in cortico-basal ganglia circuits causes specific alterations in neuronal firing that may cause disorders of the TS spectrum. Current studies using iPSC-derived basal ganglia organoids confirm thwarted development of inhibitory and cholinergic interneurons in TS and suggest potential pathophysiological mechanisms (see below).

To model disorders of brain development in a human system, the Vaccarino lab has adopted the induced pluripotent stem cell (iPSC) model and over the last several years has derived over 600 iPSC lines from patients with developmental disorders. Vaccarino and colleagues pioneered the generation of cortical organoids from iPSC lines (Mariani et al, 2012) and contributed fundamental work on neurodevelopmental alterations in severe, idiopathic ASD using this tool. Transcriptome and cell fate studies in organoids from individuals affected with ASD as compared to unaffected family members indicated alterations in cell proliferation, overproduction of synapses and a striking imbalance between inhibitory and excitatory neurons and their precursors (Mariani et al, Cell, 2015). The work has also revealed an important role of the transcription factor FOXG1 in the overproduction of GABAegic precursor cells (Mariani et al, 2015).

Other studies integrated genomes, transcriptomes and cellular phenotypes to better characterize the organoids as a model for brain development. As part of the PsychENCODE collaborative multi-site project, the lab generated a genome-scale catalog of transcripts and functional DNA elements, primarily enhancers, in iPSC-derived organoids and human brain specimens of the same genetic background. The work established that brain organoids reflect the human cerebral cortex at embryonic to early fetal stages of human brain development, and display a large repertoire of active enhancers that control neuronal fate and differentiation (Amiri et al, 2018).

Current studies are using single cell approaches to characterize brain organoids in people with different genetic background and in patients with ASD, integrating gene expression with gene regulatory elements and chromatin architecture.

In recent years, somatic mosaicism has emerged as a potential source of differences in developmental trajectories among individuals. Somatic mosaicism is the accumulation of mutations in cellular genomes after fertilization. These can be variations in DNA sequence, including single nucleotide variations (SNVs) and small insertions/deletions (Indels), or variations in DNA copy number (CNVs), in the form of large duplication/deletions. The notion that cells in an individual organism do not share the same genome is not new, but only in the last decade the scientific community has begun to use emerging deep sequencing technologies to understand the scale of this phenomenon. Somatic mutations are present in both normal cells and in various diseases (Jourdon et al, 2020) and somatic variation has been suggested to play a role in driving neuronal diversity and genome evolution. We collaborate closely with the computational genomics laboratory of Alexej Abyzov at the Mayo Clinic. Together, we have demonstrated and quantified widespread somatic mosaicism in many human cell types and tissues, including human skin fibrolasts (Abyzov et al, Nature, 2012; Abyzov et al, Genome Research, 2017; Fasching et al, Science, 2021) and the human developing brain (Bae et al, Science, 2018).

We are part of the Brain Somatic Mosaicism (BSMN) network, sponsored by the National Institute of Mental Health (NIMH) and encompassing laboratories from several major centers in the US.

Using postmortem tissue, our group was the first to use retrospective somatic mutations to delineate a lineage map for the early human embryo, which suggested that a mosaic single nucleotide variant (SNV) is generated at each cell division (rate: 1.3 SNVs/cell/mitosis) (Bae et al, Science, 2018). Since then, we determined that lineage reconstruction can be done in living persons, opening the possibility of using routine lineage reconstruction for understanding individual differences in development, as well as early diagnosis of many medical conditions involving lineage asymmetry and clonal expansion due to MM, including cancer and epilepsy (Fasching et al, Science, 2021).

Our current work seeks to develop a comprehensive yet minimally invasive approach, including experimental procedures and computational methods, for generating individualized lineage and mosaicism maps. If successful, it could provide a fundamental shift in clinical practice from considering just the individual germline genome to considering the germline genome along with the individual lineage and mosaicism maps and, perhaps, updating these maps over an individual’s lifetime.


Research Interests

Central Nervous System Diseases; Mice, Inbred Strains; Mice, Transgenic; Mosaicism; Neuroanatomy; Neurosciences; Patients; Regeneration; Developmental Biology; Cell Lineage; Living Donors; Psychiatry and Psychology; Human Genetics

Research Images

Selected Publications

Clinical Trials

ConditionsStudy Title
Child Development & AutismUnderstanding Neurodevelopment in Autism