Givens, S. E. | Search bioRxiv

bioRxiv2026

hMSCs for Osteocyte-like Cell Networks within Strain-Stiffening Bottlebrush Polymer Hydrogels

Bone formation and remodeling depend on dynamic biochemical and biomechanical signaling from the collagen-rich osteoid that precedes mineralization, yet the role of osteoid nonlinear mechanics in regulating osteocyte-like network formation remains poorly understood. Here, we engineered a synthetic bottlebrush polymer hydrogel (BB) that mimics key mechanical features of osteoid and compared it to collagen type-I (Col1) matrices with matched shear modulus (~70 Pa) and strain-stiffening behavior. Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) were cultured for 28 days in growth (GM) or osteogenic media (OM) to examine network formation, and functional connectivity using live cell fluorescence recovery after photobleaching. hMSCs cultured in BB networks and OM showed upregulation of early osteocyte markers compared to Col1. We find that strain-stiffening materials with minimal stress relaxation promote osteocyte-like cell differentiation with functional connectivity, establishing osteoid-mimetic BB hydrogels as a promising matrix to study human osteocytogenesis and osteocyte mechanotransduction in vitro.

bioengineering↗

bioRxiv2024

Healthy human induced pluripotent stem cell-derived cardiomyocytes exhibit sex dimorphism even without the addition of hormones

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a valuable cell type for studying human cardiac health and disease in vitro. However, it is not known whether hiPSC-CM display sex dimorphism and therefore whether sex should be incorporated as a biological variable in in vitro studies that include this cell type. To date, the vast majority of studies that utilize hiPSC-CM do not include both male and female sex nor stratify results based on sex because it is challenging to amass such a cohort of cells. Here we generated three female and three male hiPSC-lines from adult left ventricular cardiac fibroblasts as a resource for studying sex differences in in vitro cardiac models. We used this resource to generate hiPSC-CM and maintained them in basal media without exogenous hormones. Functional assessment of CM showed enhanced calcium handling in female-derived hiPSC-CM relative to male. Bulk RNA sequencing revealed over 300 differentially expressed genes (DEG) between male and female hiPSC-CM. Some of the DEG are X and Y-linked genes and many are implicated in cardiac health and disease including potassium channels which could account for net differences in calcium handling shown here. Gene ontology analysis of DEG showed distinct differences in pathways related to cardiac pathology including cell-cell adhesion, metabolic processes, and response to ischemic stress. These findings highlight the importance of considering sex as a variable when conducting studies to evaluate aspects of human cardiac health and disease related to cardiomyocyte function.

cell biology↗