Toward modeling the human nervous system in a dish: recent progress and outstanding challenges

Studying the cellular and molecular bases governing development, and normal and abnormal functions of the human CNS is hampered by its complexity and the very limited possibility of experimentally manipulating it in vivo. Development of 3D, tissue-like culture systems offers much promise for boosting our understanding of human neural development, birth defects, neurodegenerative diseases and neural injury, and for providing platforms that will more accurately predict efficacy of putative therapeutic compounds and assess responses to potentially neurotoxic agents. Although novel technological developments and a more interdisciplinary approach to modeling the human CNS are accelerating the pace of discovery, increasing the complexity of in vitro systems increases the ordeals to be overcome to establish highly reproducible models amenable to quantitative analysis

Cord blood Lin(-)CD45(-) embryonic-like stem cells are a heterogeneous population that lack self-renewal capacity.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Human umbilical cord blood (hUCB) has been proposed to contain not only haematopoietic stem cells, but also a rare pluripotent embryonic-like stem cell (ELSc) population that is negative for hematopoietic markers (Lin(-)CD45(-)) and expresses markers typical of pluripotent cells. The aim of this work was to isolate, characterise and expand this ELSc fraction from hUCB, as it may provide a valuable cell source for regenerative medicine applications. We found that we could indeed isolate a Lin(-)CD45(-) population of small cells (3-10 µm diameter) with a high nucleus to cytoplasm ratio that expressed the stem cell markers CD34 and CXCR4. However, in contrast to some previous reports, this fraction was not positive for CD133. Furthermore, although these cells expressed transcripts typical of pluripotent cells, such as SOX2, OCT3/4, and NANOG, they were not able to proliferate in any of the culture media known to support stem cell growth that we tested. Further analysis of the Lin(-)CD45(-) population by flow cytometry showed the presence of a Lin(-)CD45(-)Nestin(+) population that were also positive for CD34 (20%) but negative for CXCR4. These data suggest that the Lin(-)CD45(-) stem cell fraction present in the cord blood represents a small heterogeneous population with phenotypic characteristics of stem cells, including a Lin(-)CD45(-)Nestin(+) population not previously described. This study also suggests that heterogeneity within the Lin(-)CD45(-) cell fraction is the likely explanation for differences in the hUCB cell populations described by different groups that were isolated using different methods. These populations have been widely called "embryonic-like stem cell" on the basis of their phenotypical similarity to embryonic stem cells. However, the fact they do not seem to be able to self-renew casts some doubt on their identity, and warns against defining them as "embryonic-like stem cell" at this stage.Anthony Nolan and the Great Ormond Street Hospital Charity for financial support. Cesar Alvarez-Gonzalez is a fellowship from Consejo Nacional de Ciencia y Tecnologia (CONACyT) and Instituto Jaliscience de la Juventud (IJJ); Mexico

Loss of Tiparp results in aberrant layering of the cerebral cortex

Yes2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP) is an enzyme that adds a single ADP-ribose moiety to itself or other proteins. Tiparp is highly expressed in the brain; however, its function in this organ is unknown. Here, we used Tiparp–/– mice to determine Tiparp’s role in the development of the prefrontal cortex. Loss of Tiparp resulted in an aberrant organization of the mouse cortex, where the upper layers presented increased cell density in the knock-out mice compared with wild type. Tiparp loss predominantly affected the correct distribution and number of GABAergic neurons. Furthermore, neural progenitor cell proliferation was significantly reduced. Neural stem cells (NSCs) derived from Tiparp–/– mice showed a slower rate of migration. Cytoskeletal components, such as α-tubulin are key regulators of neuronal differentiation and cortical development. α-tubulin mono-ADP ribosylation (MAR) levels were reduced in Tiparp–/– cells, suggesting that Tiparp plays a role in the MAR of α-tubulin. Despite the mild phenotype presented by Tiparp–/– mice, our findings reveal an important function for Tiparp and MAR in the correct development of the cortex. Unravelling Tiparp’s role in the cortex, could pave the way to a better understanding of a wide spectrum of neurological diseases which are known to have increased expression of TIPARP.European Union Seventh Framework Program (FP7-PEOPLE-2013-COFUND) Grant n609020-Scientia Fellows (to G.G.) and by the Johan Throne Holst Foundation and the University of Oslo (J.M.)

Low temperature exposure induces browning of bone marrow stem cell derived adipocytes in vitro

Brown and beige adipocytes are characterised as expressing the unique mitochondrial uncoupling protein (UCP)1 for which the primary stimulus in vivo is cold exposure. The extent to which cold-induced UCP1 activation can also be achieved in vitro, and therefore perform a comparable cellular function, is unknown. We report an in vitro model to induce adipocyte browning using bone marrow (BM) derived mesenchymal stem cells (MSC), which relies on differentiation at 32°C instead of 37°C. The low temperature promoted browning in adipogenic cultures, with increased adipocyte differentiation and upregulation of adipogenic and thermogenic factors, especially UCP1. Cells exhibited enhanced uncoupled respiration and metabolic adaptation. Cold-exposed differentiated cells showed a marked translocation of leptin to adipocyte nuclei, suggesting a previously unknown role for leptin in the browning process. These results indicate that BM-MSC can be driven to forming beige-like adipocytes in vitro by exposure to a reduced temperature. This in vitro model will provide a powerful tool to elucidate the precise role of leptin and related hormones in hitherto functions in the browning process

Modelling healthy and injured human central nervous system using human neural stem cells in 2D and 3D cultures

The complexity of the human central nervous system (CNS), together with the limited possibility of experimentally manipulating it in vivo pose major challenges to studying human neural development, neurodegenerative diseases and responses to injury at the cellular and molecular level. The need for a reliable and readily available human CNS models can be solved by three-dimensional (3D) cell cultures that provide a more physiologically relevant environment than conventional 2D cultures. The aim of this work was to develop 3D culture models of normal and injured human CNS using human neuroblastoma cell lines and human foetal brain-derived neural stem cells (hNSCs). Characterization of hNSCs in comparison with mesenchymal stem cells highlighted differences in their immunogenicity, and led to the discovery of a novel subset of hNSCs co-expressing the neural marker SOX2 and MHC class-II antigen in vitro as well as in vivo during human CNS development. Optimal conditions for culturing undifferentiated and differentiated hNSCs in 3D differed from those reported for animal cells. hNSCs were not viable in collagen gels, but grew and differentiated in combined collagen I and/or Matrigel hydrogels. hNSCs and neuroblastoma cells showed distinct behaviour and gene expression in different matrices and in 3D as compared to 2D cultures, highlighting the importance of cell-matrix interactions and culture architecture. An in vitro hypoxia-ischemia injury model using oxygen-glucose deprivation was established in 3D cultures. Injury in these in vitro systems led to up-regulation of a cell death-associated citrullinating enzyme, peptidylarginine deiminase-3, paralleled to that observed in in vivo animal injury models. The 3D culture models developed provide novel platforms for studying human neural cell biology, including injury responses, and for neuroprotective drug testing

Modelling human CNS injury with human neural stem cells in 2- and 3-Dimensional cultures

The adult human central nervous system (CNS) has very limited regenerative capability, and injury at the cellular and molecular level cannot be studied in vivo. Modelling neural damage in human systems is crucial to identifying species-specific responses to injury and potentially neurotoxic compounds leading to development of more effective neuroprotective agents. Hence we developed human neural stem cell (hNSC) 3-dimensional (3D) cultures and tested their potential for modelling neural insults, including hypoxic-ischaemic and Ca2+-dependent injury. Standard 3D conditions for rodent cells support neuroblastoma lines used as human CNS models, but not hNSCs, but in all cases changes in culture architecture alter gene expression. Importantly, response to damage differs in 2D and 3D cultures and this is not due to reduced drug accessibility. Together, this study highlights the impact of culture cytoarchitecture on hNSC phenotype and damage response, indicating that 3D models may be better predictors of in vivo response to damage and compound toxicity

A matter of identity — Phenotype and differentiation potential of human somatic stem cells

Human somatic stem cells with neural differentiation potential can be valuable for developing cell-based therapies, including treatment of birth-related defects, while avoiding issues associated with cell reprogramming. Precisely defining the “identity” and differentiation potential of somatic stem cells from different sources, has proven difficult, given differences in sets of specific markers, protocols used and lack of side-by-side characterization of these cells in different studies. Therefore, we set to compare expression of mesenchymal and neural markers in human umbilical cord-derived mesenchymal stem cells (UC-MSCs), pediatric adipose-derived stem cells (p-ADSCs) in parallel with human neural stem cells (NSCs). We show that UC-MSCs at a basal level express mesenchymal and so-called “neural” markers, similar to that we previously reported for the p-ADSCs. All somatic stem cell populations studied, independently from tissue and patient of origin, displayed a remarkably similar expression of surface markers, with the main difference being the restricted expression of CD133 and CD34 to NSCs. Expression of certain surface and neural markers was affected by the expansion medium used. As predicted, UC-MSCs and p-ADSCs demonstrated tri-mesenchymal lineage differentiation potential, though p-ADSCs display superior chondrogenic differentiation capability. UC-MSCs and p-ADSCs responded also to neurogenic induction by up-regulating neuronal markers, but crucially they appeared morphologically immature when compared with differentiated NSCs. This highlights the need for further investigation into the use of these cells for neural therapies. Crucially, this study demonstrates the lack of simple means to distinguish between different cell types and the effect of culture conditions on their phenotype, and indicates that a more extensive set of markers should be used for somatic stem cell characterization, especially when developing therapeutic approaches