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Effects of erythropoietin in murine-induced pluripotent cell-derived panneural progenitor cells.

ABSTRACT: Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1-3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of β-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis.

SUBMITTER: Offen N

PROVIDER: S-EPMC3883967 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Effects of erythropoietin in murine-induced pluripotent cell-derived panneural progenitor cells.

Offen Nils N Flemming Johannes J Kamawal Hares H Ahmad Ruhel R Wolber Wanja W Geis Christian C Zaehres Holm H Schöler Hans R HR Ehrenreich Hannelore H Müller Albrecht M AM Sirén Anna-Leena AL

Molecular medicine (Cambridge, Mass.) 20131108

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on ...[more]

PMID: 24408113

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Project description:BackgroundCurrently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications.MethodsWe generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice.ResultsGenerated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 1018 cells per initially seeded 106 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable.ConclusionWe describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

Project description:Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation.

Dataset Information

Effects of erythropoietin in murine-induced pluripotent cell-derived panneural progenitor cells.

Publications

Effects of erythropoietin in murine-induced pluripotent cell-derived panneural progenitor cells.

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