Oliver Brüstle

In vitro differentiation of transplantable neural precursors from human embryonic stem cells

The remarkable developmental potential and replicative capacity of human embryonic stem (ES) cells promise an almost unlimited supply of specific cell types for transplantation therapies. Here we describe the in vitro differentiation, enrichment, and transplantation of neural precursor cells from human ES cells. Upon aggregation to embryoid bodies, differentiating ES cells formed large numbers of neural tube–like structures in the presence of fibroblast growth factor 2 (FGF-2). Neural precursors within these formations were isolated by selective enzymatic digestion and further purified on the basis of differential adhesion. Following withdrawal of FGF-2, they differentiated into neurons, astrocytes, and oligodendrocytes. After transplantation into the neonatal mouse brain, human ES cell–derived neural precursors were incorporated into a variety of brain regions, where they differentiated into both neurons and astrocytes. No teratoma formation was observed in the transplant recipients. These results depict human ES cells as a source of transplantable neural precursors for possible nervous system repair.

Copy number variation and selection during reprogramming to pluripotency

The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.

Direct Conversion of Fibroblasts into Stably Expandable Neural Stem Cells

Recent advances have suggested that direct induction of neural stem cells (NSCs) could provide an alternative to derivation from somatic tissues or pluripotent cells. Here we show direct derivation of stably expandable NSCs from mouse fibroblasts through a curtailed version of reprogramming to pluripotency. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming, we generated neurosphere-like colonies that could be expanded for more than 50 passages and do not depend on sustained expression of the reprogramming factors. These induced neural stem cells (iNSCs) uniformly display morphological and molecular features of NSCs, such as the expression of Nestin, Pax6, and Olig2, and have a genome-wide transcriptional profile similar to that of brain-derived NSCs. Moreover, iNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. Our results demonstrate that functional NSCs can be generated from somatic cells by factor-driven induction.

A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration

An intriguing question in human embryonic stem cell (hESC) biology is whether these pluripotent cells can give rise to stably expandable somatic stem cells, which are still amenable to extrinsic fate instruction. Here, we present a pure population of long-term self-renewing rosette-type hESC-derived neural stem cells (lt-hESNSCs), which exhibit extensive self-renewal, clonogenicity, and stable neurogenesis. Although lt-hESNSCs show a restricted expression of regional transcription factors, they retain responsiveness to instructive cues promoting the induction of distinct subpopulations, such as ventral midbrain and spinal cord fates. Using lt-hESNSCs as a donor source for neural transplantation, we provide direct evidence that hESC-derived neurons can establish synaptic connectivity with the mammalian nervous system. Combining long-term stability, maintenance of rosette-properties and phenotypic plasticity, lt-hESNSCs may serve as useful tool to study mechanisms of human NSC self-renewal, lineage segregation, and functional in vivo integration.

Sirt1 contributes critically to the redox-dependent fate of neural progenitors

Repair processes that are activated in response to neuronal injury, be it inflammatory, ischaemic, metabolic, traumatic or other cause, are characterized by a failure to replenish neurons and by astrogliosis. The underlying molecular pathways, however, are poorly understood. Here, we show that subtle alterations of the redox state, found in different brain pathologies, regulate the fate of mouse neural progenitor cells (NPCs) through the histone deacetylase (HDAC) Sirt1. Mild oxidation or direct activation of Sirt1 suppressed proliferation of NPCs and directed their differentiation towards the astroglial lineage at the expense of the neuronal lineage, whereas reducing conditions had the opposite effect. Under oxidative conditions in vitro and in vivo, Sirt1 was upregulated in NPCs, bound to the transcription factor Hes1 and subsequently inhibited pro-neuronal Mash1. In utero shRNA-mediated knockdown of Sirt1 in NPCs prevented oxidation-mediated suppression of neurogenesis and caused upregulation of Mash1 in vivo. Our results provide evidence for an as yet unknown metabolic master switch that determines the fate of neural progenitors.

Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats

Limited experimental access to the central nervous system (CNS) is a key problem in the study of human neural development, disease, and regeneration. We have addressed this problem by generating neural chimeras composed of human and rodent cells. Fetal human brain cells implanted into the cerebral ventricles of embryonic rats incorporate individually into all major compartments of the brain, generating widespread CNS chimerism. The human cells differentiate into neurons, astrocytes, and oligodendrocytes, which populate the host fore-, mid-, and hindbrain. These chimeras provide a unique model to study human neural cell migration and differentiation in a functional nervous system.

Induction of Pluripotency in Adult Unipotent Germline Stem Cells

Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.

High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity

Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb–3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.

Host-guided migration allows targeted introduction of neurons into the embryonic brain.

The stereotyped positions occupied by individual classes of neurons are a fundamental characteristic of CNS cytoarchitecture. To study the regulation of neuronal positioning, we injected genetically labeled neural precursors derived from dorsal and ventral mouse forebrain into the telencephalic vesicles of embryonic rats. Cells from both areas were found to participate in the generation of telencephalic, diencephalic, and mesencephalic brain regions. Donor-derived neurons populated the host brain in distinct patterns and acquired phenotypic features appropriate for their final location. These observations indicate that neuronal migration and differentiation are predominantly regulated by non-cell-autonomous signals. Exploiting this phenomenon, intrauterine transplantation allows generation of controlled chimerism in the mammalian brain.

Excitation-induced ataxin-3 aggregation in neurons from patients with Machado–Joseph disease

Machado–Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that l-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca2+-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na+ and K+ channels as well as ionotropic and voltage-gated Ca2+ channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.