Neural cells differentiated in vitro from human embryonic stem cells (hESC) exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. Here, we describe ...standard conditions for the use and discovery of markers for analysis and cell selection of hESC undergoing neuronal differentiation. To generate better‐defined cell populations, we established a working protocol for sorting heterogeneous hESC‐derived neural cell populations by fluorescence‐activated cell sorting (FACS). Using genetically labeled synapsin‐green fluorescent protein‐positive hESC‐derived neurons as a proof of principle, we enriched viable differentiated neurons by FACS. Cell sorting methodology using surface markers was developed, and a comprehensive profiling of surface antigens was obtained for immature embryonic stem cell types (such as stage‐specific embryonic antigen SSEA‐3, ‐4, TRA‐1‐81, TRA‐1‐60), neural stem and precursor cells (such as CD133, SSEA‐1 CD15, A2B5, forebrain surface embryonic antigen‐1, CD29, CD146, p75 CD271), and differentiated neurons (such as CD24 or neural cell adhesion molecule NCAM; CD56). At later stages of neural differentiation, NCAM (CD56) was used to isolate hESC‐derived neurons by FACS. Such FACS‐sorted hESC‐derived neurons survived in vivo after transplantation into rodent brain. These results and concepts provide (a) a feasible approach for experimental cell sorting of differentiated neurons, (b) an initial survey of surface antigens present during neural differentiation of hESC, and (c) a framework for developing cell selection strategies for neural cell‐based therapies.
Disclosure of potential conflicts of interest is found at the end of this article.
It is currently not known whether dopamine (DA) neurons derived from human embryonic stem cells (hESCs) can survive in vivo and alleviate symptoms in models of Parkinson disease (PD). Here, we report ...the use of Noggin (a bone morphogenic protein antagonist) to induce neuroectodermal cell development and increase the yield of DA neurons from hESCs. A combination of stromal‐derived inducing activity and Noggin markedly enhanced the generation of neuroepithelial progenitors that could give rise to DA neurons. In addition, Noggin diminished the occurrence of a fibroblast‐like Nestin‐positive precursor population that differentiated into myocytes. After transplantation of differentiated hESCs to a rodent model of PD, some grafts contained human midbrain‐like DA neurons. This protocol demonstrates hESC derivation and survival of human DA neurons appropriate for cell therapy in PD.
Abstract
The development of cell-, gene- and tissue engineering (CGT)-based therapies must adhere to strict pharmaceutical quality management standards, as for any other biological or small-molecule ...drug. However, early developments often failed to fully comply with good laboratory practices (GLP) in non-clinical safety studies. Despite an upward trend of positive opinions in marketing authorization applications, evidence of adherence to the principles of GLP is not openly reported; therefore, their relative impact on the overall quality of the product development program is unknown. Herein we investigated the actual degree of GLP implementation and the underlying factors impeding full compliance in non-clinical developments of CGT-based marketed medicines in the EU and USA, including (i) the co-existence of diverse quality management systems of more strategic value for small organizations, particularly current Good Manufacturing Practices n(GMP); (ii) lack of regulatory pressure to pursue GLP certification; and (iii) the involvement of public institutions lacking a pharmaceutical mindset and resources. As a final reflection, we propose conformity to good research practice criteria not as a doctrinaire impediment to scientific work, but as a facilitator of efficient clinical translation of more effective and safer innovative therapies.
Chimeras for the twenty-first century Morata Tarifa, Cynthia; López Navas, Luis; Azkona, Garikoitz ...
Critical reviews in biotechnology,
04/2020, Letnik:
40, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Recent advances in stem cell biology and molecular engineering have improved and simplified the methodology employed to create experimental chimeras, highlighting their value in basic research and ...broadening the spectrum of potential applications. Experimental chimeras have been used for decades during the generation of murine genetic models, this being especially relevant in developmental and regeneration studies. Indeed, their value for the research and modeling of human diseases was recognized by the 2007 Nobel Prize to Mario Capecchi, Martin Evans, and Oliver Smithies. More recently, their potential application in regenerative medicine has generated a lot of interest, particularly the enticing possibility to generate human organs for transplantation in livestock animals. In this review, we provide an update on interspecific chimeric organogenesis, its possibilities, current limitations, alternatives, and ethical issues.
Although implantation of fetal dopamine (DA) neurons can reduce parkinsonism in patients, current methods are rudimentary, and a reliable donor cell source is lacking. We show that transplanting low ...doses of undifferentiated mouse embryonic stem (ES) cells into the rat striatum results in a proliferation of ES cells into fully differentiated DA neurons. ES cell-derived DA neurons caused gradual and sustained behavioral restoration of DA-mediated motor asymmetry. Behavioral recovery paralleled in vivo positron emission tomography and functional magnetic resonance imaging data demonstrating DA-mediated hemodynamic changes in the striatum and associated brain circuitry. These results demonstrate that transplanted ES cells can develop spontaneously into DA neurons. Such DA neurons can restore cerebral function and behavior in an animal model of Parkinson's disease.
In preterm birth germinal matrix hemorrhages (GMHs) and the consequent posthemorrhagic hydrocephalus (PHH), the neuroepithelium/ependyma development is disrupted. This work is aimed to explore the ...possibilities of ependymal repair in GMH/PHH using a combination of neural stem cells, ependymal progenitors (EpPs), and mesenchymal stem cells.
GMH/PHH was induced in 4-day-old mice using collagenase, blood, or blood serum injections. PHH severity was characterized 2 weeks later using magnetic resonance, immunofluorescence, and protein expression quantification with mass spectrometry. Ependymal restoration and wall regeneration after stem cell treatments were tested in vivo and in an ex vivo experimental approach using ventricular walls from mice developing moderate and severe GMH/PHH. The effect of the GMH environment on EpP differentiation was tested in vitro. Two-tailed Student
or Wilcoxon-Mann-Whitney
test was used to find differences between the treated and nontreated groups. ANOVA and Kruskal-Wallis tests were used to compare >2 groups with post hoc Tukey and Dunn multiple comparison tests, respectively.
PHH severity was correlated with the extension of GMH and ependymal disruption (means, 88.22% severe versus 19.4% moderate). GMH/PHH hindered the survival rates of the transplanted neural stem cells/EpPs. New multiciliated ependymal cells could be generated from transplanted neural stem cells and more efficiently from EpPs (15% mean increase). Blood and TNFα (tumor necrosis factor alpha) negatively affected ciliogenesis in cells committed to ependyma differentiation (expressing Foxj1 forkhead box J1 transcription factor). Pretreatment with mesenchymal stem cells improved the survival rates of EpPs and ependymal differentiation while reducing the edematous (means, 18% to 0.5% decrease in severe edema) and inflammatory conditions in the explants. The effectiveness of this therapeutical strategy was corroborated in vivo (means, 29% to 0% in severe edema).
In GMH/PHH, the ependyma can be restored and edema decreased from either neural stem cell or EpP transplantation in vitro and in vivo. Mesenchymal stem cell pretreatment improved the success of the ependymal restoration.
•Rats showed reduced performance in behavior tests as they aged.•Aged rats had high death rates during training and after injury and cell transplantation.•Neural progenitor cells were present in the ...spinal cord 4 weeks after transplantation.•Neural progenitor cells could migrate and differentiate and did not cause tumors.•Aged rats failed to recover locomotor activity one month after cell transplantation.
With an expanding elderly population, an increasing number of older adults will experience spinal cord injury (SCI) and might be candidates for cell-based therapies, yet there is a paucity of research in this age group. The objective of the present study was to analyze how aged rats tolerate behavioral testing, surgical procedures, post-operative complications, intra-spinal cell transplantation and immunosuppression, and to examine the effectiveness of human iPSC-derived Neural Progenitor Cells (IMR90-hiPSC-NPCs) in a model of SCI. We performed behavioral tests in rats before and after inducing cervical hemi-contusions at C4 level with a fourth-generation Ohio State University Injury Device. Four weeks later, we injected IMR90-hiPSC-NPCs in animals that were immunosuppressed by daily cyclosporine injection. Four weeks after injection we analyzed locomotor behavior and mortality, and histologically assessed the survival of transplanted human NPCs. As rats aged, their success at completing behavioral tests decreased. In addition, we observed high mortality rates during behavioral training (41.2%), after cervical injury (63.2%) and after cell injection (50%). Histological analysis revealed that injected cells survived and remained at and around the grafted site and did not cause tumors. No locomotor improvement was observed in animals four weeks after IMR90-hiPSC-NPC transplantation. Our results show that elderly rats are highly vulnerable to interventions, and thus large groups of animals must be initially established to study the potential efficacy of cell-based therapies in age-related chronic myelopathies.
Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both familial and idiopathic forms of Parkinson's disease (PD). Neuroinflammation is a key event in neurodegeneration and aging, and ...there is mounting evidence of LRRK2 involvement in inflammatory pathways. In a previous study, we described an alteration of the inflammatory response in dermal fibroblasts from PD patients expressing the G2019S and R1441G mutations in LRRK2.
Taking advantage of cellular reprogramming, we generated induced pluripotent stem cell (iPSC) lines and neurons thereafter, harboring LRRK2
and LRRK2
mutations. We used gene silencing and functional reporter assays to characterize the effect of the mutations. We examined the temporal profile of TNFα-induced changes in proteins of the NF-κB pathway and optimized western blot analysis to capture α-synuclein dynamics. The effects of the mutations and interventions were analyzed by two-way ANOVA tests with respect to corresponding controls.
LRRK2 silencing decreased α-synuclein protein levels in mutated neurons and modified NF-κB transcriptional targets, such as PTGS2 (COX-2) and TNFAIP3 (A20). We next tested whether NF-κB and α-synuclein pathways converged and found that TNFα modulated α-synuclein levels, although we could not detect an effect of LRRK2 mutations, partly because of the individual variability. Nevertheless, we confirmed NF-κB dysregulation in mutated neurons, as shown by a protracted recovery of IκBα and a clear impairment in p65 nuclear translocation in the LRRK2 mutants.
Altogether, our results show that LRRK2 mutations affect α-synuclein regulation and impair NF-κB canonical signaling in iPSC-derived neurons. TNFα modulated α-synuclein proteostasis but was not modified by the LRRK2 mutations in this paradigm. These results strengthen the link between LRRK2 and the innate immunity system underscoring the involvement of inflammatory pathways in the neurodegenerative process in PD.
Dopamine (DA) neurons can be derived from human and primate embryonic stem (ES) cells in vitro. An ES cell–based replacement therapy for patients with Parkinson's disease requires that in ...vitro–generated neurons maintain their phenotype in vivo. Other critical issues relate to their proliferative capacity and risk of tumor formation, and the capability of migration and integration in the adult mammalian brain. Neural induction was achieved by coculture of primate parthenogenetic ES cells (Cyno‐1) with stromal cells, followed by sequential exposure to midbrain patterning and differentiation factors to favor DA phenotypic specification. Differentiated ES cells were treated with mitomycin C and transplanted into adult immunosuppressed rodents and into a primate (allograft) with out immunosuppression. A small percentage of DA neurons survived in both rodent and primate hosts for the entire term of the study (4 and 7 months, respectively). Other neuronal and glial populations derived from Cyno‐1 ES cells showed, in vivo, phenotypic characteristics and growth and migration patterns similar to fetal primate transplants, and a majority of cells (>80%) expressed the forebrain transcription factor brain factor 1. No teratoma formation was observed. In this study, we demonstrate long‐term survival of DA neurons obtained in vitro from primate ES cells. Optimization of differentiation, cell selection, and cell transfer is required for functional studies of ES‐derived DA neurons for future therapeutic applications.
Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the ...disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.
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