Induced neural stem cells achieve long-term survival and functional integration in the adult mouse brain.

Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications.

Origin-dependent neural cell identities in differentiated human iPSCs in vitro and after transplantation into the mouse brain.

The differentiation capability of induced pluripotent stem cells (iPSCs) toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs), dermal fibroblasts, or cord blood CD34(+) hematopoietic progenitor cells. Neural progenitor cells (NPCs) and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.

Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling.

Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.

A systemic transcriptome analysis reveals the regulation of neural stem cell maintenance by an E2F1-miRNA feedback loop.

Stem cell fate decisions are controlled by a molecular network in which transcription factors and miRNAs are of key importance. To systemically investigate their impact on neural stem cell (NSC) maintenance and neuronal commitment, we performed a high-throughput mRNA and miRNA profiling and isolated functional interaction networks of involved mechanisms. Thereby, we identified an E2F1-miRNA feedback loop as important regulator of NSC fate decisions. Although E2F1 supports NSC proliferation and represses transcription of miRNAs from the miR-17∼92 and miR-106a∼363 clusters, these miRNAs are transiently up-regulated at early stages of neuronal differentiation. In these early committed cells, increased miRNAs expression levels directly repress E2F1 mRNA levels and inhibit cellular proliferation. In mice, we demonstrated that these miRNAs are expressed in the neurogenic areas and that E2F1 inhibition represses NSC proliferation. The here presented data suggest a novel interaction mechanism between E2F1 and miR-17∼92 / miR-106a∼363 miRNAs in controlling NSC proliferation and neuronal differentiation.

Induced pluripotent stem cells generated from adult bone marrow-derived cells of the nonhuman primate (Callithrix jacchus) using a novel quad-cistronic and excisable lentiviral vector.

Regenerative medicine is in need of solid, large animal models as a link between rodents and humans to evaluate the functionality, immunogenicity, and clinical safety of stem cell-derived cell types. The common marmoset (Callithrix jacchus) is an excellent large animal model, genetically close to humans and readily used worldwide in clinical research. Until now, only two groups showed the generation of induced pluripotent stem cells (iPSCs) from the common marmoset using integrating retroviral vectors. Therefore, we reprogrammed bone marrow-derived mesenchymal cells (MSCs) of adult marmosets in the presence of TAV, SB431542, PD0325901, and ascorbic acid via a novel, excisable lentiviral spleen focus-forming virus (SFFV)-driven quad-cistronic vector system (OCT3/4, KLF4, SOX2, C-MYC). Endogenous pluripotency markers like OCT3/4, KLF4, SOX2, C-MYC, LIN28, NANOG, and strong alkaline phosphatase signals were detected. Exogenous genes were silenced and additionally the cassette was removed with a retroviral Gag precursor system. The cell line could be cultured in absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) and could be successfully differentiated into embryoid bodies and teratomas with presence of all three germ layers. Directed differentiation generated neural progenitors, megakaryocytes, adipocytes, chondrocytes, and osteogenic cells. Thus, all criteria for fully reprogrammed bone marrow-MSCs of a nonhuman primate with a genetically sophisticated construct could be demonstrated. These cells will be a promising tool for future autologous transplantations.

Anti-inflammatory treatment induced regenerative oligodendrogenesis in parkinsonian mice.

INTRODUCTION: The adult mammalian brain retains niches for neural stem cells (NSCs), which can generate glial and neuronal components of the brain tissue. However, it is barely established how chronic neuroinflammation, as it occurs in neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, affects adult neurogenesis and, therefore, modulates the brain's potential for self-regeneration. METHODS: Neural stem cell culture techniques, intraventricular tumor necrosis factor (TNF)-α infusion and the 6-hydroxydopamine mouse model were used to investigate the influence of neuroinflammation on adult neurogenesis in the Parkinson's disease background. Microscopic methods and behavioral tests were used to analyze samples. RESULTS: Here, we demonstrate that differences in the chronicity of TNF-α application to cultured NSCs result in opposed effects on their proliferation. However, chronic TNF-α treatment, mimicking Parkinson's disease associated neuroinflammation, shows detrimental effects on neural progenitor cell activity. Inversely, pharmacological inhibition of neuroinflammation in a 6-hydroxydopamine mouse model led to increased neural progenitor cell proliferation in the subventricular zone and neuroblast migration into the lesioned striatum. Four months after surgery, we measured improved Parkinson's disease-associated behavior, which was correlated with long-term anti-inflammatory treatment. But surprisingly, instead of newly generated striatal neurons, oligodendrogenesis in the striatum of treated mice was enhanced. CONCLUSIONS: We conclude that anti-inflammatory treatment, in a 6-hydroxydopamine mouse model for Parkinson's disease, leads to activation of adult neural stem cells. These adult neural stem cells generate striatal oligodendrocytes. The higher numbers of newborn oligodendrocytes possibly contribute to axonal stability and function in this mouse model of Parkinson's disease and thereby attenuate dysfunctions of basalganglian motor-control.

Direct reprogramming of fibroblasts into neural stem cells by defined factors.

Recent studies have shown that defined sets of transcription factors can directly reprogram differentiated somatic cells to a different differentiated cell type without passing through a pluripotent state, but the restricted proliferative and lineage potential of the resulting cells limits the scope of their potential applications. Here we show that a combination of transcription factors (Brn4/Pou3f4, Sox2, Klf4, c-Myc, plus E47/Tcf3) induces mouse fibroblasts to directly acquire a neural stem cell identity-which we term as induced neural stem cells (iNSCs). Direct reprogramming of fibroblasts into iNSCs is a gradual process in which the donor transcriptional program is silenced over time. iNSCs exhibit cell morphology, gene expression, epigenetic features, differentiation potential, and self-renewing capacity, as well as in vitro and in vivo functionality similar to those of wild-type NSCs. We conclude that differentiated cells can be reprogrammed directly into specific somatic stem cell types by defined sets of specific transcription factors.