Microgravity, Stem Cells, and Embryonic Development: Challenges and Opportunities for 3D Tissue Generation

Space is a challenging environment for the human body, due to the combined effects of reduced gravity (microgravity) and cosmic radiation. Known effects of microgravity range from the blood redistribution that affects the cardiovascular system and the eye to muscle wasting, bone loss, anemia, and immune depression. About cosmic radiation, the shielding provided by the spaceship hull is far less efficient than that afforded at ground level by the combined effects of the Earth atmosphere and magnetic field. The eye and its nervous layer (the retina) are affected by both microgravity and heavy ions exposure. Considering the importance of sight for long-term manned flights, visual research aimed at devising measures to protect the eye from environmental conditions of the outer space represents a special challenge to meet. In this review we focus on the impact of microgravity on embryonic development, discussing the roles of mechanical forces in the context of the neutral buoyancy the embryo experiences in the womb. At variance with its adverse effects on the adult human body, simulated microgravity may provide a unique tool for understanding the biomechanical events involved in the development and assembly in vitro of three-dimensional (3D) ocular tissues. Prospective benefits are the development of novel safety measures to protect the human eye from cosmic radiation in microgravity during long-term manned spaceflights in the outer space, as well as the generation of human 3D-retinas with its supporting structures to develop innovative and effective therapeutic options for degenerative eye diseases

GABAergic Neuronal Precursor Grafting: Implications in Brain Regeneration and Plasticity

Numerous neurological disorders are caused by a dysfunction of the GABAergic system that impairs or either stimulates its inhibitory action over its neuronal targets. Pharmacological drugs have generally been proved very effective in restoring its normal function, but their lack of any sort of spatial or cell type specificity has created some limitations in their use. In the last decades, cell-based therapies using GABAergic neuronal grafts have emerged as a promising treatment, since they may restore the lost equilibrium by cellular replacement of the missing/altered inhibitory neurons or modulating the hyperactive excitatory system. In particular, the discovery that embryonic ganglionic eminence-derived GABAergic precursors are able to disperse and integrate in large areas of the host tissue after grafting has provided a strong rationale for exploiting their use for the treatment of diseased brains. GABAergic neuronal transplantation not only is efficacious to restore normal GABAergic activities but can also trigger or sustain high neuronal plasticity by promoting the general reorganization of local neuronal circuits adding new synaptic connections. These results cast new light on dynamics and plasticity of adult neuronal assemblies and their associated functions disclosing new therapeutic opportunities for the near future

Direct Neuronal Reprogramming Reveals Unknown Functions for Known Transcription Factors

In recent years, the need to derive sources of specialized cell types to be employed for cell replacement therapies and modeling studies has triggered a fast acceleration of novel cell reprogramming methods. In particular, in neuroscience, a number of protocols for the efficient differentiation of somatic or pluripotent stem cells have been established to obtain a renewable source of different neuronal cell types. Alternatively, several neuronal populations have been generated through direct reprogramming/transdifferentiation, which concerns the conversion of fully differentiated somatic cells into induced neurons. This is achieved through the forced expression of selected transcription factors (TFs) in the donor cell population. The reprogramming cocktail is chosen after an accurate screening process involving lists of TFs enriched into desired cell lineages. In some instances, this type of studies has revealed the crucial role of TFs whose function in the differentiation of a given specific cell type had been neglected or underestimated. Herein, we will speculate on how the in vitro studies have served to better understand physiological mechanisms of neuronal development in vivo

Microgravity, Stem Cells, and Embryonic Development: Challenges and Opportunities for 3D Tissue Generation

Space is a challenging environment for the human body, due to the combined effects of reduced gravity (microgravity) and cosmic radiation. Known effects of microgravity range from the blood redistribution that affects the cardiovascular system and the eye to muscle wasting, bone loss, anemia, and immune depression. About cosmic radiation, the shielding provided by the spaceship hull is far less efficient than that afforded at ground level by the combined effects of the Earth atmosphere and magnetic field. The eye and its nervous layer (the retina) are affected by both microgravity and heavy ions exposure. Considering the importance of sight for long-term manned flights, visual research aimed at devising measures to protect the eye from environmental conditions of the outer space represents a special challenge to meet. In this review we focus on the impact of microgravity on embryonic development, discussing the roles of mechanical forces in the context of the neutral buoyancy the embryo experiences in the womb. At variance with its adverse effects on the adult human body, simulated microgravity may provide a unique tool for understanding the biomechanical events involved in the development and assembly in vitro of three-dimensional (3D) ocular tissues. Prospective benefits are the development of novel safety measures to protect the human eye from cosmic radiation in microgravity during long-term manned spaceflights in the outer space, as well as the generation of human 3D-retinas with its supporting structures to develop innovative and effective therapeutic options for degenerative eye diseases

Generation of two human iPSC lines, FINCBi002-A and FINCBi003-A, carrying heteroplasmic macrodeletion of mitochondrial DNA causing Pearson's syndrome

Pearson marrow pancreas syndrome (PMPS) is a sporadic mitochondrial disease, resulting from the clonal expansion of a mutated mitochondrial DNA (mtDNA) molecule bearing a macro-deletion, and therefore missing essential genetic information. PMPS is characterized by the presence of deleted (Δ) mtDNA that co-exist with the presence of a variable amount of wild-type mtDNA, a condition termed heteroplasmy. All tissues of the affected individual, including the haemopoietic system and the post-mitotic, highly specialized tissues (brain, skeletal muscle, and heart) contain the large-scale mtDNA deletion in variable amount. We generated human induced pluripotent stem cells (hiPSCs) from two PMPS patients, carrying different type of large-scale deletion

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell–derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration

Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery

P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifi

Generation of a human iPSC line, FINCBi001-A, carrying a homoplasmic m.G3460A mutation in MT-ND1 associated with Leber's Hereditary optic Neuropathy (LHON)

Leber's Hereditary Optic Neuropathy (LHON) is a maternally inherited disorder caused by homoplasmic mutations of mitochondrial DNA (mtDNA). LHON is characterized by the selective degeneration of the retinal ganglion cells (RGC). Almost all LHON maternal lineages are homoplasmic mutant (100% mtDNA copies are mutant) for one of three frequent mtDNA mutations now found in over 90% of patients worldwide (m.11778G > A/MT-ND4, m.3460G > A/MT-ND1, m.14484 T > C/MT-ND6). Human induced pluripotent stem cells (hiPSCs) were generated from a patient carrying the homoplasmic m.3460G > A/MT-ND1 mutation using the Sendai virus non-integrating virus

A Human Stem Cell-Derived Neurosensory-Epithelial Circuitry on a Chip to Model Herpes Simplex Virus Reactivation

Both emerging viruses and well-known viral pathogens endowed with neurotropism can either directly impair neuronal functions or induce physio-pathological changes by diffusing from the periphery through neurosensory-epithelial connections. However, developing a reliable and reproducible in vitro system modeling the connectivity between the different human sensory neurons and peripheral tissues is still a challenge and precludes the deepest comprehension of viral latency and reactivation at the cellular and molecular levels. This study shows a stable topographic neurosensory-epithelial connection on a chip using human stem cell-derived dorsal root ganglia (DRG) organoids. Bulk and single-cell transcriptomics showed that different combinations of key receptors for herpes simplex virus 1 (HSV-1) are expressed by each sensory neuronal cell type. This neuronal-epithelial circuitry enabled a detailed analysis of HSV infectivity, faithfully modeling its dynamics and cell type specificity. The reconstitution of an organized connectivity between human sensory neurons and keratinocytes into microfluidic chips provides a powerful in vitro platform for modeling viral latency and reactivation of human viral pathogens

Defective Neurogenesis in Citron Kinase Knockout Mice by Altered Cytokinesis and Massive Apoptosis

AbstractCitron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K−/− mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS