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Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Thermo Fisher Scientific

The proneural protein neurogenin 2 (NGN2) is a key transcription factor in regulating both neurogenesis and neuronal radial migration in the embryonic cerebral cortex. However, the co-factors that support the action of NGN2 in the cortex remain unclear. Here, we show that the LIM-only protein LMO4 functions as a novel co-factor of NGN2 in the developing cortex. LMO4 and its binding partner nuclear LIM interactor (NLI/LDB1/CLIM2) interact with NGN2 simultaneously, forming a multi-protein transcription complex. This complex is recruited to the E-box containing enhancers of NGN2-target genes, which regulate various aspects of cortical development, and activates NGN2-mediated transcription. Correspondingly, analysis of Lmo4-null embryos shows that the loss of LMO4 leads to impairments of neuronal differentiation in the cortex. In addition, expression of LMO4 facilitates NGN2-mediated radial migration of cortical neurons in the embryonic cortex. Our results indicate that LMO4 promotes the acquisition of cortical neuronal identities by forming a complex with NGN2 and subsequently activating NGN2-dependent gene expression.

LMO4 functions as a co-activator of neurogenin 2 in the developing cortex.

An excitingly predictable 'omic future.

Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones.

CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming.

Pluripotent stem cells (PSCs) are powerful tools for basic scientific research and promising agents for drug discovery and regenerative medicine. Technological advances have made it increasingly easy to generate PSCs but the various lines generated may differ in their characteristics based on their origin, derivation, number of passages, and culture conditions. In order to confirm the pluripotency, quality, identity, and safety of pluripotent cell lines as they are derived and maintained, it is critical to perform a panel of characterization assays. Functional pluripotency is determined using tests that rely on the expression of specific markers in the undifferentiated and differentiated states; tests for quality, identity and safety are less specialized. This article provides a comprehensive review of current practices in PSC characterization and explores challenges in the field, from the selection of markers to the development of simple and scalable methods. It also delves into emerging trends like the adoption of alternative assays that could be used to supplement or replace traditional methods, specifically the use of in silico assays for determining pluripotency.

Joanna Asprer

Life Sciences Solutions

Thermo Fisher Scientific

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Joanna Asprer

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Thermo Fisher Scientific

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Life Sciences Solutions