Establishment of an induced pluripotent stem cell (iPSC) line from a 9-year old male with autism spectrum disorder (ASD).

Peripheral blood mononuclear cells (PBMCs) were collected from a clinically characterized patient with autism spectrum disorder (ASD). The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The pluripotency of transgene-free iPSCs was verified by immunocytochemistry for pluripotency markers and by spontaneous in vitro differentiation towards the 3 germ layers. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of ASD, also for drug testing, early biomarker discovery and gene therapy studies.

Establishment of EHMT1 mutant induced pluripotent stem cell (iPSC) line from a 11-year-old Kleefstra syndrome (KS) patient with autism and normal intellectual performance.

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous, de novo, premature termination codon (PTC) mutation (NM_024757.4(EHMT1):c.3413G>A; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome, also for drug testing, early biomarker discovery and gene therapy studies.

Grafted murine induced pluripotent stem cells prevent death of injured rat motoneurons otherwise destined to die.

Human plexus injuries often include the avulsion of one or more ventral roots, resulting in debilitating conditions. In this study the effects of undifferentiated murine iPSCs on damaged motoneurons were investigated following avulsion of the lumbar 4 (L4) ventral root, an injury known to induce the death of the majority of the affected motoneurons. Avulsion and reimplantation of the L4 ventral root (AR procedure) were accompanied by the transplantation of murine iPSCs into the injured spinal cord segment in rats. Control animals underwent ventral root avulsion and reimplantation, but did not receive iPSCs. The grafted iPSCs induced an improved reinnervation of the reimplanted ventral root by the host motoneurons as compared with the controls (number of retrogradely labeled motoneurons: 503 ± 38 [AR+iPSCs group] vs 48 ± 6 [controls, AR group]). Morphological reinnervation resulted in a functional recovery, i.e. the grafted animals exhibited more motor units in their reinnervated hind limb muscles, which produced a greater force than that in the controls (50 ± 2.1% vs 11.9 ± 4.2% maximal tetanic tension [% ratio of operated/intact side]). Grafting of undifferentiated iPSCs downregulated the astroglial activation within the L4 segment. The grafted cells differentiated into neurons and astrocytes in the injured cord. The grafted iPSCs, host neurons and glia were found to produce the cytokines and neurotrophic factors MIP-1a, IL-10, GDNF and NT-4. These findings suggest that, following ventral root avulsion injury, iPSCs are able to induce motoneuron survival and regeneration through combined neurotrophic and cytokine modulatory effects.

N-cadherin negatively regulates collective Drosophila glial migration through actin cytoskeleton remodeling.

Cell migration is an essential and highly regulated process. During development, glia cells and neurons migrate over long distances - in most cases collectively - to reach their final destination and build the sophisticated architecture of the nervous system, the most complex tissue of the body. Collective migration is highly stereotyped and efficient, defects in the process leading to severe human diseases that include mental retardation. This dynamic process entails extensive cell communication and coordination, hence, the real challenge is to analyze it in the entire organism and at cellular resolution. We here investigate the impact of the N-cadherin adhesion molecule on collective glial migration, by using the Drosophila developing wing and cell-type specific manipulation of gene expression. We show that N-cadherin timely accumulates in glial cells and that its levels affect migration efficiency. N-cadherin works as a molecular brake in a dosage-dependent manner, by negatively controlling actin nucleation and cytoskeleton remodeling through α/ß catenins. This is the first in vivo evidence for N-cadherin negatively and cell autonomously controlling collective migration.

Interlocked loops trigger lineage specification and stable fates in the Drosophila nervous system.

Multipotent precursors are plastic cells that generate different, stable fates at the correct number, place and time, to allow tissue and organ formation. While fate determinants are known to trigger specific transcriptional programs, the molecular pathway driving the progression from multipotent precursors towards stable and specific identities remains poorly understood. Here we demonstrate that, in Drosophila neural precursors, the glial determinant glial cell missing (Gcm) acts as a 'time bomb' and triggers its own degradation once the glial programme is stably activated. This requires a sequence of transcriptional and posttranscriptional loops, whereby a Gcm target first affects the expression and then acetylation of the fate determinant, thus controlling Gcm levels and stability over time. Defective homeostasis between the loops alters the neuron:glia ratio and freezes cells in an intermediate glial/neuronal phenotype. In sum, we identify an efficient strategy triggering cell identity, a process altered in pathological conditions such as cancer.

Tissue resident stem cells: till death do us part.

Aging is accompanied by reduced regenerative capacity of all tissues and organs and dysfunction of adult stem cells. Notably, these age-related alterations contribute to distinct pathophysiological characteristics depending on the tissue of origin and function and thus require special attention in a type by type manner. In this paper, we review the current understanding of the mechanisms leading to tissue-specific adult stem cell dysfunction and reduced regenerative capacity with age. A comprehensive investigation of the hematopoietic, the neural, the mesenchymal, and the skeletal stem cells in age-related research highlights that distinct mechanisms are associated with the different types of tissue stem cells. The link between age-related stem cell dysfunction and human pathologies is discussed along with the challenges and the future perspectives in stem cell-based therapies in age-related diseases.

Generation of neuronal progenitor cells and neurons from mouse sleeping beauty transposon-generated induced pluripotent stem cells.

Mouse embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells can be used as models of neuronal differentiation for the investigation of mammalian neurogenesis, pharmacological testing, and development of cell-based therapies. Recently, mouse iPS cell lines have been generated by Sleeping Beauty (SB) transposon-mediated transgenesis (SB-iPS). In this study, we determined for the first time the differentiation potential of mouse SB-iPS cells to form neuronal progenitor cells (NPCs) and neurons. Undifferentiated SB-iPS and ES cells were aggregated into embryoid bodies (EBs) and cultured in neuronal differentiation medium supplemented with 5 µM all-trans retinoic acid. Thereafter, EBs were dissociated and plated to observe further neuronal differentiation. Samples were fixed on days 10 and 14 for immunocytochemistry staining using the NPC markers Pax6 and Nestin and the neuron marker ßIII-tubulin/Tuj1. Nestin-labeled cells were analyzed further by flow cytometry. Our results demonstrated that SB-iPS cells can generate NPCs and differentiate further into neurons in culture, although SB-iPS cells produced less nestin-positive cells than ESCs (6.12 ± 1.61 vs. 74.36 ± 1.65, respectively). In conclusion, the efficiency of generating SB-iPS cells-derived NPCs needs to be improved. However, given the considerable potential of SB-iPS cells for drug testing and as therapeutic models in neurological disorders, continuing investigation of their neuronal differentiation ability is required.

Homeostatic interactions at the front of migration control the integrity and the efficiency of a migratory glial chain.

In metazoans, cell migration often occurs in a collective manner: the cells move while physically and functionally connected to their neighbors. The coordinated and timely movement of the cells eventually ensures the proper organization of tissues, and deregulation in such a process contributes to the development of severe diseases. Thus, understanding the cellular mechanisms underlying coordinated cell movement is of great interest in basic and medical science. The developing Drosophila wing provides an excellent model to follow the chain migration of glial cells in vivo. Cells at the tip of the glial collective have been shown to control the timely movement of the chain. In the present study, we show that while pioneers trigger chain migration, they cannot move as single cells. We also show that isolating cell clusters at the chain tip restores the formation of smaller migratory communities. Interestingly, the migratory efficiency of these de novo formed communities depends on the number of cells and progressively improves as the size of the cluster increases. Thus, homeostatic events at the migratory front control community integrity, efficiency, and coordination, emphasizing the importance of interactions and cell counting in fine-tuning collective processes.

Astroglia genesis in vitro: distinct effects of retinoic acid in different phases of neural stem cell differentiation.

In the developing CNS, the manifestation of the macro-glial phenotypes is delayed behind the formation of neurons. The "neurons first--glia second" principle seems to be valid for neural tissue differentiation throughout the neuraxis, but the reasons behind are far from clear. In the presented study, the mechanisms of this timing were investigated in vitro, in the course of the neural differentiation of one cell derived NE-4C neuroectodermal stem and P19 embryonic carcinoma cells. The data demonstrated that astrocyte formation coincided in time with the maturation of postmitotic neurons, but the close vicinity of neurons did not initiate astrocyte formation before schedule. All-trans retinoic acid, a well-known inducer of neuronal differentiation, on the other hand, blocked effectively the astroglia production if present in defined stages of the in vitro neuroectodermal cell differentiation. According to the data, retinoic acid plays at least a dual role in astrogliogenesis: while it is needed for committing neural progenitors for a future production of astrocytes, it prevents premature astrogliogenesis by inhibiting the differentiation of primed glial progenitors.