Metabolic Switch Under Glucose Deprivation Leading to Discovery of NR2F1 as a Stimulus of Osteoblast Differentiation.

Poor survival of grafted cells is the major impediment of successful cell-based therapies for bone regeneration. Implanted cells undergo rapid death in an ischemic environment largely because of hypoxia and metabolic stress from glucose deficiency. Understanding the intracellular metabolic processes and finding genes that can improve cell survival in these inhospitable conditions are necessary to enhance the success of cell therapies. Thus, the purpose of this study was to investigate changes of metabolic profile in glucose-deprived human bone marrow stromal/stem cells (hBMSCs) through metabolomics analysis and discover genes that could promote cell survival and osteogenic differentiation in a glucose-deprived microenvironment. Metabolomics analysis was performed to determine metabolic changes in a glucose stress metabolic model. In the absence of glucose, expression levels of all metabolites involved in glycolysis were significantly decreased than those in a glucose-supplemented state. In glucose-deprived osteogenic differentiation, reliance on tricarboxylic acid cycle (TCA)-predicted oxidative phosphorylation instead of glycolysis as the main mechanism for energy production in osteogenic induction. By comparing differentially expressed genes between glucose-deprived and glucose-supplemented hBMSCs, NR2F1 (Nuclear Receptor Subfamily 2 Group F Member 1) gene was discovered to be associated with enhanced survival and osteogenic differentiation in cells under metabolic stress. Small, interfering RNA (siRNA) for NR2F1 reduced cell viability and osteogenic differentiation of hBMSCs under glucose-supplemented conditions whereas NR2F1 overexpression enhanced osteogenic differentiation and cell survival of hBMSCs in glucose-deprived osteogenic conditions via the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) pathway. NR2F1-transfected hBMSCs significantly enhanced new bone formation in a critical size long-bone defect of rats compared with control vector-transfected hBMSCs. In conclusion, the results of this study provide an understanding of the metabolic profile of implanted cells in an ischemic microenvironment and demonstrate that NR2F1 treatment may overcome this deprivation by enhancing AKT and ERK regulation. These findings can be utilized in regenerative medicine for bone regeneration. © 2022 American Society for Bone and Mineral Research (ASBMR).

Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI.

Transcription factors with gradients of expression in neocortical progenitors give rise to distinct motor and sensory cortical areas by controlling the area-specific differentiation of distinct neuronal subtypes. However, the molecular mechanisms underlying this area-restricted control are still unclear. Here, we show that COUP-TFI controls the timing of birth and specification of corticospinal motor neurons (CSMN) in somatosensory cortex via repression of a CSMN differentiation program. Loss of COUP-TFI function causes an area-specific premature generation of neurons with cardinal features of CSMN, which project to subcerebral structures, including the spinal cord. Concurrently, genuine CSMN differentiate imprecisely and do not project beyond the pons, together resulting in impaired skilled motor function in adult mice with cortical COUP-TFI loss-of-function. Our findings indicate that COUP-TFI exerts critical areal and temporal control over the precise differentiation of CSMN during corticogenesis, thereby enabling the area-specific functional features of motor and sensory areas to arise.

Altered molecular regionalization and normal thalamocortical connections in cortex-specific Pax6 knock-out mice.

Transcription factor Pax6 exerts a prominent rostrolateral(high) to caudomedial(low) expression gradient in the cortical progenitors and have been implicated in regulation of area identity in the mammalian cortex. Herein, we analyzed the role of Pax6 in molecular arealization and development of thalamocortical connections in the juvenile cortex-specific conditional Pax6 knock-out mice (Pax6cKO). Using a set of molecular markers of positional identity (Id2, Cadherin6, COUP-TF1, RZRbeta, and EphA7), we show that, in the juvenile Pax6cKO, the relative size of caudal cortical areas (putative visual and somatosensory) are mildly enlarged, whereas the rostral domain (putative motor) is severely reduced. Despite the rostral shift of graded expression of areal markers, the distribution of area-specific thalamocortical and corticofugal projections appear normal in the Pax6cKO. This indicates that change of the size of cortical areas is not accompanied by a change in cortical identity. We show furthermore that, despite a severe depletion of supragranular cortical layers and accumulation of cells along the pallial-subpallial boundary, thalamocortical fibers establish a periphery-related pattern of the somatosensory cortex in normal position in Pax6cKO. Our findings indicate that Pax6 expression gradients in cortical progenitors do not directly impart thalamocortical or corticofugal areal identity.