Serena G. Giannelli

Direct Conversion of Fibroblasts into Functional Astrocytes by Defined Transcription Factors

Summary Direct cell reprogramming enables direct conversion of fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell-lineage-specific transcription factors. This approach is rapid and simple, generating the cell types of interest in one step. However, it remains unknown whether this technology can be applied to convert fibroblasts into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis, and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB, and SOX9 to be sufficient to convert with high efficiency embryonic and postnatal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene-expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications.

Adult human Müller glia cells are a highly efficient source of rod photoreceptors.

There is growing evidence that Müller glia cells (MGCs) might act as regenerative elements in injured retinas of fishes and amniotes. However, their differentiation potential in humans is yet unknown. We isolated Müller glia from adult human retinas and propagated them in vitro revealing for the first time their ability to differentiate into rod photoreceptors. These results were also confirmed with mice retinas. Here, we describe conditions by which human MGCs adopt a rod photoreceptor commitment with a surprising efficiency as high as 54%. Functional characterization of Müller glia‐derived photoreceptors by patch‐clamp recordings revealed that their electrical properties are comparable to those of adult rods. Interestingly, our procedure allowed efficient derivation of MGC cultures starting from both injured and degenerating and postmortem human retinas. Human transplanted Müller glia‐derived photoreceptors integrate and survive within immunodeficient mouse retinas. These data provide evidence that Müller glia retains an unpredicted plasticity and multipotent potential into adulthood, and it is therefore a promising source of novel therapeutic applications in retinal repair. STEM CELLS 2011;29:344–356

Rapid Conversion of Fibroblasts into Functional Forebrain GABAergic Interneurons by Direct Genetic Reprogramming

Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders.

Remote control of induced dopaminergic neurons in parkinsonian rats.

Direct lineage reprogramming through genetic-based strategies enables the conversion of differentiated somatic cells into functional neurons and distinct neuronal subtypes. Induced dopaminergic (iDA) neurons can be generated by direct conversion of skin fibroblasts; however, their in vivo phenotypic and functional properties remain incompletely understood, leaving their impact on Parkinsons disease (PD) cell therapy and modeling uncertain. Here, we determined that iDA neurons retain a transgene-independent stable phenotype in culture and in animal models. Furthermore, transplanted iDA neurons functionally integrated into host neuronal tissue, exhibiting electrically excitable membranes, synaptic currents, dopamine release, and substantial reduction of motor symptoms in a PD animal model. Neuronal cell replacement approaches will benefit from a system that allows the activity of transplanted neurons to be controlled remotely and enables modulation depending on the physiological needs of the recipient; therefore, we adapted a DREADD (designer receptor exclusively activated by designer drug) technology for remote and real-time control of grafted iDA neuronal activity in living animals. Remote DREADD-dependent iDA neuron activation markedly enhanced the beneficial effects in transplanted PD animals. These data suggest that iDA neurons have therapeutic potential as a cell replacement approach for PD and highlight the applicability of pharmacogenetics for enhancing cellular signaling in reprogrammed cell-based approaches.

Direct Derivation of Neural Rosettes from Cloned Bovine Blastocysts: A Model of Early Neurulation Events and Neural Crest Specification In Vitro

Embryonic stem cells differentiate into neuroectodermal cells under specific culture conditions. In primates, these cells are organized into rosettes expressing Pax6 and Sox1 and are responsive to inductive signals such as Sonic hedgehog (Shh) and retinoic acid. However, direct derivation of organized neuroectoderm in vitro from preimplantation mammalian embryos has never been reported. Here, we show that bovine inner cell masses from nuclear transfer and fertilized embryos, grown on feeders in serum‐free medium, form polarized rosette structures expressing nestin, Pax6, Pax7, Sox1, and Otx2 and exhibiting interkinetic nuclear migration activity and cell junction distribution as in the developing neural tube. After in vitro expansion, neural rosettes give rise to p75‐positive neural crest precursor cell lines capable of long‐term proliferation and differentiation in autonomic and sensory peripheral neurons, glial cells, melanocytes, smooth muscle cells, and chondrocytes, recapitulating in vitro the unique plasticity of the neural crest lineage. Challenging the rosette dorsal fate by early exposure to Shh induces the expression of ventral markers Isl1, Nkx2.2, and Nkx6.1 and differentiation of mature astrocytes and neurons of central nervous system ventral identity, demonstrating appropriate response to inductive signals. All together, these findings indicate that neural rosettes directly derived from cloned and fertilized bovine embryos represent an in vitro model of early neural specification and differentiation events. Moreover, this study provides a source of highly proliferative neural crest precursor cell lines of wide differentiation potential for cell therapy and tissue engineering applications.

Embryonic stem-derived versus somatic neural stem cells: A comparative analysis of their developmental potential and molecular phenotype

Reliable procedures to induce neural commitment of totipotent undifferentiated embryonic stem (ES) cells have provided new tools for investigating the molecular mechanisms underlying cell fate choices. We extensively characterized the developmental potential of ES‐induced neural cells obtained using an adaptation of the multistep induction protocol. We provided evidence that ES‐derived neural proliferating cells are endowed with stem cell properties such as extensive self‐renewal capacity and single‐cell multipotency. In differentiating conditions, cells matured exclusively into neurons, astrocytes, and oligodendrocytes. All these features have been previously described in only somatic neural stem cells (NSCs). Therefore, we consider it more appropriate to rename our cells ES‐derived NSCs. These similarities between the two NSC populations induced us to carefully compare their proliferation ability and differentiation potential. Although they were very similar in overall behavior, we scored specific differences. For instance, ES‐derived NSCs proliferated at higher rate and consistently generated a higher number of neurons compared with somatic NSCs. To further investigate their relationships, we carried out a molecular analysis comparing their transcriptional profiles during proliferation. We observed a large fraction of shared expressed transcripts, including genes previously described to be critical in defining somatic NSC traits. Among the genes differently expressed, candidate genes possibly responsible for divergences between the two cell types were selected and further investigated. In particular, we showed that an enhanced MAPK (mitogen‐activated protein kinase) signaling is acting in ES‐induced NSCs, probably triggered by insulin‐like growth factor–II. This may contribute to the high proliferation rate exhibited by these cells in culture.

Long-term culture and differentiation of CNS precursors derived from anterior human neural rosettes following exposure to ventralizing factors

In this study we demonstrated that neural rosettes derived from human ES cells can give rise either to neural crest precursors, following expansion in presence of bFGF and EGF, or to dopaminergic precursors after exposure to ventralizing factors Shh and FGF8. Both regionalised precursors are capable of extensive proliferation and differentiation towards the corresponding terminally differentiated cell types. In particular, peripheral neurons, cartilage, bone, smooth muscle cells and also pigmented cells were obtained from neural crest precursors while tyrosine hydroxylase and Nurr1 positive dopaminergic neurons were derived from FGF8 and Shh primed rosette cells. Gene expression and immunocytochemistry analyses confirmed the expression of dorsal and neural crest genes such as Sox10, Slug, p75, FoxD3, Pax7 in neural precursors from bFGF-EGF exposed rosettes. By contrast, priming of rosettes with FGF8 and Shh induced the expression of dopaminergic markers Engrailed1, Pax2, Pitx3, floor plate marker FoxA2 and radial glia markers Blbp and Glast, the latter in agreement with the origin of dopaminergic precursors from floor plate radial glia. Moreover, in vivo transplant of proliferating Shh/FGF8 primed precursors in parkinsonian rats demonstrated engraftment and terminal dopaminergic differentiation. In conclusion, we demonstrated the derivation of long-term self-renewing precursors of selected regional identity as potential cell reservoirs for cell therapy applications, such as CNS degenerative diseases, or for the development of toxicological tests.

Modeling physiological and pathological human neurogenesis in the dish.

New advances in directing the neuronal differentiation of human embryonic and induced pluripotent stem cells (hPSCs, abbreviation intended to convey both categories of pluripotent stem cells) have promoted the development of culture systems capable of modeling early neurogenesis and neural specification at some of their critical milestones. The hPSC-derived neural rosette can be considered the in vitro counterpart of the developing neural tube, since both structures share a virtually equivalent architecture and related functional properties. Epigenetic stimulation methods can modulate the identity of the rosette neural progenitors in order to generate authentic neuronal subtypes, as well as a full spectrum of neural crest derivatives. The intrinsic capacity of induced pluripotent cell-derived neural tissue to self-organize has become fully apparent with the emergence of innovative in vitro systems that are able to shape the neuronal differentiation of hPSCs into organized tissues that develop in three dimensions. However, significant hurdles remain that must be completely solved in order to facilitate the use of hPSCs in modeling (e.g., late-onset disorders) or in building therapeutic strategies for cell replacement. In this direction, new procedures have been established to promote the maturation and functionality of hPSC-derived neurons. Meanwhile, new methods to accelerate the aging of in vitro differentiating cells are still in development. hPSC-based technology has matured enough to offer a significant and reliable model system for early and late neurogenesis that could be extremely informative for the study of the physiological and pathological events that occur during this process. Thus, full exploitation of this cellular system can provide a better understanding of the physiological events that shape human brain structures, as well as a solid platform to investigate the pathological mechanisms at the root of human diseases.

Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy

Allogeneic fetal‐derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient‐specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene‐therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC‐derived neural stem cells (NSCs) showing a reliable “NSC signature” is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS‐NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a “gold standard” in a side‐by‐side comparison when validating the phenotype of hiPS‐NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS‐NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA‐overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA‐overexpressing iPSC‐derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient‐specific ARSA‐overexpressing hiPS‐NSCs may be used in autologous ex vivo gene therapy protocols to provide long‐lasting enzymatic supply in MLD‐affected brains. Stem Cells Translational Medicine 2017;6:352–368

COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons

COUP-TFI is an orphan nuclear receptor acting as a strong transcriptional regulator in different aspects of forebrain embryonic development. In this study, we investigated COUP-TFI expression and function in the mouse olfactory bulb (OB), a highly plastic telencephalic region in which continuous integration of newly generated inhibitory interneurons occurs throughout life. OB interneurons belong to different populations that originate from distinct progenitor lineages. Here, we show that COUP-TFI is highly expressed in tyrosine hydroxylase (TH)-positive dopaminergic interneurons in the adult OB glomerular layer (GL). We found that odour deprivation, which is known to downregulate TH expression in the OB, also downregulates COUP-TFI in dopaminergic cells, indicating a possible correlation between TH- and COUP-TFI-activity-dependent action. Moreover, we demonstrate that conditional inactivation of COUP-TFI in the EMX1 lineage results in a significant reduction of both TH and ZIF268 expression in the GL. Finally, lentiviral vector-mediated COUP-TFI deletion in adult-generated interneurons confirmed that COUP-TFI acts cell-autonomously in the control of TH and ZIF268 expression. These data indicate that COUP-TFI regulates TH expression in OB cells through an activity-dependent mechanism involving ZIF268 induction and strongly argue for a maintenance rather than establishment function of COUP-TFI in dopaminergic commitment. Our study reveals a previously unknown role for COUP-TFI in the adult brain as a key regulator in the control of sensory-dependent plasticity in olfactory dopaminergic neurons.