Kinesin Light Chain 1 Suppression Impairs Human Embryonic Stem Cell Neural Differentiation and Amyloid Precursor Protein Metabolism

The etiology of sporadic Alzheimer disease (AD) is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aβ) and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aβ and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC) expressing reduced levels of the kinesin light chain 1 (KLC1) Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aβ, supporting the previously established connection between KLC1, Tau and Aβ. Intriguingly, KLC1-suppressed neural precursors (NPs), isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s) of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases

Cell-Surface Marker Signatures for the Isolation of Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells

Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations

Human neural cultures with reduced KLC1 exhibit altered APP metabolism.

<p>(A) APP proteolytic processing by either β-and γ-secretases or α- and γ-secretases produces sAPPβ and Aβ (shaded dark grey) or sAPPα and p3 fragments, respectively. (B–C) PA6 feeder neural differentiation cultures were harvested after seven weeks and equal protein from control and <i>shKLC1-1</i> cultures were analyzed using Western blots. (B) Representative immunoblots for full length APP in control and <i>shKLC1-1</i> neural differentiation lysates. Results for both amino (APP-N; LN27) and carboxy terminal (APP-C) antibodies are shown. The APP carboxyl terminal cleavage fragments were not reliably detected. (C) Quantification of full length APP levels relative to NSE. (D) Levels of extracellular human Aβ peptides 38, 40 or 42 amino acids in length detected in media conditioned by control or <i>shKLC1-1</i> hESC co-cultured with PA6 feeder cells for seven weeks. Human Aβ was not detected from PA6 feeder only cultures. (E) Levels of Triton X-100 soluble intracellular human Aβ-40 in control or <i>shKLC1-1</i> PA6 feeder differentiation cultures aged seven weeks. Intracellular Aβ peptides 38 or 42 amino acids long were not detectable. (F) Levels of human extracellular sAPPα and sAPPβ were detected in media conditioned by control or <i>shKLC1-1</i> PA6 feeder cocultures aged <i>in vitro</i> for seven weeks. Based on n = 6 each line; *p<0.05, **p<0.01 by 2-tailed t-test.</p

Undifferentiated KLC1-suppressed hESC exhibit normal morphology, pluripotency marker expression and karyotypes.

<p>(A) Representative images of control, <i>shKLC1-1</i> and <i>shKLC1-2</i> undifferentiated hESC cultures showing bordered colony morphology typical of pluripotent cells (arrows). Scale bar 200 micrometers. (B) Immunofluorescence staining of KLC1 in undifferentiated hESC control, <i>shKLC1-1</i> and <i>shKLC1-2</i> colonies. Merged images show overlay of KLC1 (red) and DAPI-stained nuclei (blue). Scale bar 50 micrometers. (C) Equal protein from undifferentiated control, <i>shKLC1-1</i> and <i>shKLC-2</i> culture lysates were analyzed by Western blot for KLC1 and Actin. Bar graph shows Actin normalized KLC1 levels relative to control. n = 3; **p<0.01, ***p<0.001 by 2-tailed t-test compared to control. (D–E) Immunofluorescence imagesof undifferentiated control, <i>shKLC1-1</i> and <i>shKLC1-2</i> cultures for pluripotency markers Oct-4 (D) and TRA-1-81 (E). Merged images show overlay of Oct-4 (D; red) or TRA-1-81 (E; red) and DAPI-stained nuclei (blue). Scale bar 50 micrometers. (F) Bivariate plots show distribution of cells in control, <i>shKLC1-1</i> and <i>shKLC1-2</i> undifferentiated cultures Oct-4+TRA-1-81+ (in blue). Data is representative of three experiments.</p

Neural cultures derived from KLC1-suppressed hESC have reduced neural microtubule-associated markers.

<p>(A) Cultures were harvested at seven weeks <i>in vitro</i> and equal protein from mouse PA6 feeders cultured with control, <i>shKLC1-1</i> or no hESC (PA6 feeder cells lane) were analyzed by Western blotting for actin, GAPDH and SOD1. Note that unlike Actin, mouse and human GAPDH and SOD1 have different electrophoretic mobilities (arrows). (B–C) Control or <i>shKLC1-1</i> hESC were cultured for seven weeks with PA6 feeder cells and then harvested. Equal protein from control and <i>shKLC1-1</i> cultures was analyzed by Western blotting. (B) Representative immunoblots of Actin, NSE, GFAP, α-Tubulin, β-III-Tubulin, MAP2, pNF-H, pNF-M, Tau and pTau. (C) Quantification of protein levels relative to control and normalized to Actin. Based on n = 6 wells each *p<0.05, **p<0.01, ***p<0.001 by 2 tailed t-test compared to control.</p

KLC1 and Kinesin-1C subunits are reduced in neural cultures derived from KLC1-suppressed hESC.

<p>(A) Control, <i>shKLC1-1</i> and <i>shKLC1-2</i> hESC were differentiated for seven weeks using the PA6 feeder method. Representative bright field images of control, <i>shKLC1-1</i> and <i>shKLC1-2</i> PA6 feeder cocultures collected at nine, eighteen, twenty-two and forty-eight days after plating. Arrows point to rosettes. Insets show close-ups of indicated rosettes. Arrowheads denote axon-like projections emanating from hESC derived cell clusters. Scale bars: 200 µm for main images, 50 µm for insets. (B) PA6 neural differentiation cultures were harvested after seven weeks <i>in vitro</i> and equal protein from control, <i>shKLC1-1</i> and <i>shKLC1-2</i> cultures analyzed by Western blotting for KLC1, Kinesin-1C and Actin. Bar graphs show relative quantification of KLC1 and Kinesin-1C levels relative to Actin. Based on n = 7 control and <i>shKLC1-1</i>; n = 3 <i>shKLC1-2</i>, ***p<0.001 by two-tailed Student's t-test compared to control.</p

Neural induction cultures made from KLC1-suppressed hESC have reduced cell densities and proportions of NPs.

<p>(A–D) Control, hESC were subjected to neural induction conditions for eighteen days using PA6 feeder or EB methods as indicated. (A) Bright field image of control EB neural induction culture (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029755#pone-0029755-g002" target="_blank">Figure 2A</a> for image of PA6 differentiation). Arrows point to rosettes. Insets show close-ups of indicated rosettes. Scale bars: 200 µm for main images, 50 µm for insets. (B) Percent of cells in control EB and control PA6 feeder differentiation cultures with CD184^hiCD24^hiCD44^loCD271^lo NP cell surface marker signature (C) Quantification of cell density in EB control, <i>shKLC1-1</i> and <i>shKLC1-2</i> hESC EB neural induction cultures. EB cultures were dissociated enzymatically and counted using a hemocytometer. (D). Percent of cells within EB control, <i>shKLC1-1</i> and <i>shKLC1-2</i> hESC differentiation cultures exhibiting CD184^hiCD24^hiCD44^loCD271^lo NP cell surface marker signature after. For (B–C), control n = 9, <i>shKLC1-1</i> n = 6, <i>shKLC1-2</i> n = 3. For (D), n = 3 each line. **p<0.01, ***p<0.001 by 2-tailed t-test compared to control.</p