Combined 3D bioprinting and tissue-specific ECM system reveals the influence of brain matrix on stem cell differentiation.

We have previously shown that human and murine breast extracellular matrix (ECM) can significantly impact cellular behavior, including stem cell fate determination. It has been established that tissue-specific extracellular matrix from the central nervous system has the capacity to support neuronal survival. However, the characterization of its influence on stem cell differentiation and its adaptation to robust 3D culture models is underdeveloped. To address these issues, we combined our 3D bioprinter with hydrogels containing porcine brain extracellular matrix (BMX) to test the influence of the extracellular matrix on stem cell differentiation. Our 3D bioprinting system generated reproducible 3D neural structures derived from mouse embryonic stem cells (mESCs). We demonstrate that the addition of BMX preferentially influences 3D bioprinted mESCs towards neural lineages compared to standard basement membrane (Geltrex/Matrigel) hydrogels alone. Furthermore, we demonstrate that we can transplant these 3D bioprinted neural cellular structures into a mouse's cleared mammary fat pad, where they continue to grow into larger neural outgrowths. Finally, we demonstrate that direct injection of human induced pluripotent stem cells (hiPSCS) and neural stem cells (NSCs) suspended in pure BMX formed neural structures in vivo. Combined, these findings describe a unique system for studying brain ECM/stem cell interactions and demonstrate that BMX can direct pluripotent stem cells to differentiate down a neural cellular lineage without any additional specific differentiation stimuli.

Salivary microRNA as a prospective tool for concussion diagnosis and management: A scoping review.

BACKGROUND: Despite increased efforts directed toward research, concussions are a growing concern and can be a complex injury for healthcare professionals to manage. Current practices are largely dependent on patients self-reporting symptoms and a clinical assessment, which uses objective tools that lack effectiveness. With the demonstrated effects of concussions, it is imperative that a more valid or reliable objective tool, like a clinical biomarker, be identified to improve outcomes. One potential biomarker that has shown promise is salivary microRNA. However, there is no objective consensus as to which microRNA offers the most clinical value regarding concussions, hence this review. Therefore, the purpose of this scoping review was to identify salivary miRNAs associated with concussions. METHODS: Two independent reviewers performed a literature search to identify research articles. Studies using human subjects, collected salivary miRNA, and were published in English were included. Data of interest were salivary miRNA, collection timing, and relation to concussion diagnosis or management. RESULTS: This paper reviews nine studies that analyzed salivary miRNA for concussion diagnosis and management. CONCLUSIONS: In total, the studies have identified 49 salivary miRNA that show promise in assisting with concussion practices. With continued research, the use of salivary miRNA may enhance clinicians' abilities to diagnose and manage concussions.

3D bioprinted mammary organoids and tumoroids in human mammary derived ECM hydrogels.

The extracellular matrix (ECM) of tissues is an important mediator of cell function. Moreover, understanding cellular dynamics within their specific tissue context is also important for developmental biology, cancer research, and regenerative medicine. However, robust in vitro models that incorporate tissue-specific microenvironments are lacking. Here we describe a novel mammary-specific culture protocol that combines a self-gelling hydrogel comprised solely of ECM from decellularized rat or human breast tissue with the use of our previously described 3D bioprinting platform. We initially demonstrate that undigested and decellularized mammary tissue can support mammary epithelial and tumor cell growth. We then describe a methodology for generating mammary ECM extracts that can spontaneously gel to form hydrogels. These ECM hydrogels retain unique structural and signaling profiles that elicit differential responses when normal mammary and breast cancer cells are cultured within them. Using our bioprinter, we establish that we can generate large organoids/tumoroids in the all mammary-derived hydrogel. These findings demonstrate that our system allows for growth of organoids/tumoroids in a tissue-specific matrix with unique properties, thus providing a suitable platform for ECM and epithelial/cancer cell studies. STATEMENT OF SIGNIFICANCE: Factors within extracellular matrices (ECMs) are specific to their tissue of origin. It has been shown that tissue specific factors within the mammary gland's ECM have pronounced effects on cellular differentiation and cancer behavior. Understanding the role of the ECM in controlling cell fate has major implications for developmental biology, tissue engineering, and cancer therapy. However, in vitro models to study cellular interactions with tissue specific ECM are lacking. Here we describe the generation of 3D hydrogels consisting solely of human or mouse mammary ECM. We demonstrate that these novel 3D culture substrates can sustain large 3D bioprinted organoid and tumoroid formation. This is the first demonstration of an all mammary ECM culture system capable of sustaining large structural growths.

3D bioprinter applied picosecond pulsed electric fields for targeted manipulation of proliferation and lineage specific gene expression in neural stem cells.

OBJECTIVE: Picosecond pulse electric fields (psPEF) have the potential to elicit functional changes in mammalian cells in a non-contact manner. Such electro-manipulation of pluripotent and multipotent cells could be a tool in both neural interface and tissue engineering. Here, we describe the potential of psPEF in directing neural stem cells (NSCs) gene expression, metabolism, and proliferation. As a comparison mesenchymal stem cells (MSCs) were also tested. APPROACH: A psPEF electrode was anchored on a customized commercially available 3D printer, which allowed us to deliver pulses with high spatial precision and systematically control the electrode position in three-axes. When the electrodes are continuously energized and their position is shifted by the 3D printer, large numbers of cells on a surface can be exposed to a uniform psPEF. With two electric field strengths (20 and 40 kV cm-1), cell responses, including cell viability, proliferation, and gene expression assays, were quantified and analyzed. MAIN RESULTS: Analysis revealed both NSCs and MSCs showed no significant cell death after treatments. Both cell types exhibited an increased metabolic reduction; however, the response rate for MSCs was sensitive to the change of electric field strength, but for NSCs, it appeared independent of electric field strength. The change in proliferation rate was cell-type specific. MSCs underwent no significant change in proliferation whereas NSCs exhibited an electric field dependent response with the higher electric field producing less proliferation. Further, NSCs showed an upregulation of glial fibrillary acidic protein (GFAP) after 24 h to 40 kV cm-1, which is characteristic of astrocyte specific differentiation. SIGNIFICANCE: Changes in cell metabolism, proliferation, and gene expression after picosecond pulsed electric field exposure are cell type specific.

Epigenetic alterations mediate iPSC-induced normalization of DNA repair gene expression and TNR stability in Huntington's disease cells.

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disorder caused by a cytosine-adenine-guanine (CAG) trinucleotide repeat (TNR) expansion within the HTT gene. The mechanisms underlying HD-associated cellular dysfunction in pluripotency and neurodevelopment are poorly understood. We had previously identified downregulation of selected DNA repair genes in HD fibroblasts relative to wild-type fibroblasts, as a result of promoter hypermethylation. Here, we tested the hypothesis that hypomethylation during cellular reprogramming to the induced pluripotent stem cell (iPSC) state leads to upregulation of DNA repair genes and stabilization of TNRs in HD cells. We sought to determine how the HD TNR region is affected by global epigenetic changes through cellular reprogramming and early neurodifferentiation. We find that early stage HD-affected neural stem cells (HD-NSCs) contain increased levels of global 5-hydroxymethylation (5-hmC) and normalized DNA repair gene expression. We confirm TNR stability is induced in iPSCs, and maintained in HD-NSCs. We also identify that upregulation of 5-hmC increases ten-eleven translocation 1 and 2 (TET1/2) protein levels, and show their knockdown leads to a corresponding decrease in the expression of select DNA repair genes. We further confirm decreased expression of TET1/2-regulating miR-29 family members in HD-NSCs. Our findings demonstrate that mechanisms associated with pluripotency induction lead to a recovery in the expression of select DNA repair gene and stabilize pathogenic TNRs in HD.