Manipulating Stem Cell Fate with Disordered Bioactive Cues on Surfaces: The Role of Bioactive Ligand Selection.

The development of 2D or 3D bioactive platforms for rapidly isolating pure populations of cells from adult stem cells holds promise for advancing the understanding of cellular mechanisms, drug testing, and tissue engineering. Over the years, methods have emerged to synthesize bioactive micro- and nanostructured 2D materials capable of directing stem cell fate. We introduce a novel method for randomly micro- or nanopatterning any protein/peptide onto both 2D and 3D scaffolds via spray technology. Our goal is to investigate the impact of arranging bioactive micropatterns (ordered vs disordered) on surfaces to guide human mesenchymal stem cell (hMSC) differentiation. The spray technology efficiently coats materials with controlled, cost-effective bioactive micropatterns in various sizes and shapes. BMP-2 mimetic peptides were covalently grafted, individually or in combination with RGD peptides, onto activated polyethylene terephthalate (PET) surfaces through a spraying process, incorporating nano/microscale parameters like size, shape, and composition. The study explores different peptide distributions on surfaces and various peptide combinations. Four surfaces were homogeneously functionalized with these peptides (M1 to M4 with various densities of peptides), and six surfaces with disordered micro- and nanopatterns of peptides (S0 to S5 with different sizes of peptide patterns) were synthesized. Fluorescence microscopy assessed peptide distribution, followed by hMSC culture for 2 weeks, and evaluated osteogenic differentiation via immunocytochemistry and RT-qPCR for osteoblast and osteocyte markers. Cells on uniformly peptide-functionalized surfaces exhibited cuboidal forms, while those on surfaces with disordered patterns tended toward columnar or cuboidal shapes. Surfaces S4 and S5 showed dendrite-like formations resembling an osteocyte morphology. S5 showed significant overexpression of osteoblast (OPN) and osteocyte markers (E11, DMP1, and SOST) compared to control surfaces and other micropatterned surfaces. Notably, despite sharing an equivalent quantity of peptides with a homogeneous functionalized surface, S5 displayed a distinct distribution of peptides, resulting in enhanced osteogenic differentiation of hMSCs.

Osteocyte gene expression analysis in mouse bone: optimization of a laser-assisted microdissection protocol.

Among bone cells, osteocytes are the most abundant, but also the most challenging to study because they are located inside a dense mineralized matrix. Due to their involvement in bone homeostasis, diverse tools are needed to understand their roles in bone physiology and pathology. This work was aimed at establishing a laser-assisted microdissection protocol to isolate osteocytes and analyze their gene expressions. The goal was to overcome the limitations of the technique currently most used: RNA extraction from the whole bone. To perform laser microdissection and subsequent gene expression analysis, the five main steps of the protocol have been adapted for the bone tissue. After testing many parameters, we found that the best options were (1) take unfixed snap-frozen tissue, (2) cryosection with a supported tape system to improve the tissue morphology if necessary, (3) microdissect regions of interest, and (4) recover the bone pieces by catapulting, if feasible, or by gravity. Finally, RNA extraction (5) was the most efficient with a precipitation method and allowed quantifying the expression of well described osteocyte genes (Gja1/Cx43, Phex, Pdpn, Dmp1, Sost). This work describes two protocols optimized for femur and calvaria and gives an overview of the many optimization options that one could try when facing difficulties with laser microdissection.

In vivo bioluminescence imaging of the intracerebral fibroin-controlled AAV-α-synuclein diffusion for monitoring the central nervous system and peripheral expression.

Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.

Single cell tracing of Pomc neurons reveals recruitment of 'Ghost' subtypes with atypical identity in a mouse model of obesity.

The hypothalamus contains a remarkable diversity of neurons that orchestrate behavioural and metabolic outputs in a highly plastic manner. Neuronal diversity is key to enabling hypothalamic functions and, according to the neuroscience dogma, it is predetermined during embryonic life. Here, by combining lineage tracing of hypothalamic pro-opiomelanocortin (Pomc) neurons with single-cell profiling approaches in adult male mice, we uncovered subpopulations of 'Ghost' neurons endowed with atypical molecular and functional identity. Compared to 'classical' Pomc neurons, Ghost neurons exhibit negligible Pomc expression and are 'invisible' to available neuroanatomical approaches and promoter-based reporter mice for studying Pomc biology. Ghost neuron numbers augment in diet-induced obese mice, independent of neurogenesis or cell death, but weight loss can reverse this shift. Our work challenges the notion of fixed, developmentally programmed neuronal identities in the mature hypothalamus and highlight the ability of specialised neurons to reversibly adapt their functional identity to adult-onset obesogenic stimuli.

The CB1 cannabinoid receptor regulates autophagy in the tibialis anterior skeletal muscle in mice

The endocannabinoid system (ECS) regulates energy metabolism, has been implicated in the pathogenesis of metabolic diseases and exerts its actions mainly through the type 1 cannabinoid receptor (CB1). Likewise, autophagy is involved in several cellular processes. It is required for the normal development of muscle mass and metabolism, and its deregulation is associated with diseases. It is known that the CB1 regulates signaling pathways that control autophagy, however, it is currently unknown whether the ECS could regulate autophagy in the skeletal muscle of obese mice. This study aimed to investigate the role of the CB1 in regulating autophagy in skeletal muscle. We found concomitant deregulation in the ECS and autophagy markers in high-fat diet-induced obesity. In obese CB1-KO mice, the autophagy-associated protein LC3 II does not accumulate when mTOR and AMPK phosphorylation levels do not change. Acute inhibition of the CB1 with JD-5037 decreased LC3 II protein accumulation and autophagic flux. Our results suggest that the CB1 regulates autophagy in the tibialis anterior skeletal muscle in both lean and obese mice.