Induced pluripotent stem cells (iPSCs) have become the most promising candidates for in vitro modeling of motor neuron (MN) diseases, such as amyotrophic lateral sclerosis (ALS), and possibly for ...future therapeutic implementation in regenerative medicine. We here present for the first time the differentiation of human cord-blood-derived iPSCs (hCBiPSCs) into MNs, the cell type primarily affected in ALS. In contrast to iPSCs generated from adult tissue, the hCBiPSCs used in this study hold the promise of lower genetic mutation burden or epigenetic alterations, which makes them ideal candidates for transplantation studies. Small-molecule-derived neural precursor cells (smNPCs) were generated from hCBiPSCs and used for the following differentiation studies to substantially shorten MN differentiation time. Consequently, as early as 18 days of in vitro differentiation, the MNs stained positive for neuronal- and for MN-specific markers accompanied by respective gene expression patterns. To demonstrate that the hCBiPSC-derived neural precursor cells (smNPCs) can be differentiated into functional MNs, the cells were characterized by calcium imaging and patch-clamp analysis. Calcium imaging detected the expression of functional voltage-dependent calcium and ligand-gated channels of several important neurotransmitters. Using whole-cell patch-clamp recordings, we observed functional neuronal properties like sodium-inward currents and action potentials (APs). Some cells showed spontaneous APs and synaptic activity that are signs of essential functional maturation. Having established a rapid and efficient method to generate functional MNs from hCBiPSCs, we demonstrate the differentiation potential of genetically unbiased hCBiPSCs as promising source for transplantation studies and also create a framework for future in-vitro disease modeling.
Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the ...nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS.
In response to GM-CSF or M-CSF, macrophages (MΦ) can acquire
pro- or anti-inflammatory properties, respectively. Given
the importance of CD14 and Toll-like receptor (TLR) 4 in
lipopolysaccharide ...(LPS)-induced signaling, we studied the
effect of anti-CD14 antibody mediated CD14 blockade on
LPS-induced cytokine production, signal transduction and
on the expression levels of CD14 and TLR4 in GM-MΦ and
M-MΦ. We found M-MΦ to express higher levels of both surface
antigens and to produce more interferon (IFN)-β and
interleukin-10, but less tumor necrosis factor (TNF)-α than
GM-MΦ. Blockage of CD14 at high LPS concentrations increased
the production of proinflammatory cytokines and
decreased that of IFN-β in M-MΦ but not in GM-MΦ. We
show that phosphorylation states of signaling molecules of
the MyD88 (myeloid differentiation primary response 88),
TRIF (TIR-domain-containing adapter-inducing IFN-β) and
MAPK (mitogen-activated protein kinase) pathways are not
altered in any way that would account for the cytokine overshoot
reaction. However, CD14 blockage in M-MΦ decreased
TLR4 and CD14 expression levels, regardless of the presence
of LPS, indicating that the loss of the surface molecules prevented
LPS from initiating TRIF signaling. As TNF-α synthesis
was even upregulated under these experimental conditions,
we suggest that TRIF is normally involved in restricting LPSinduced
TNF-α overproduction. Thus, surface CD14 plays a
decisive role in the biological response by determining LPSinduced
signaling.
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