Mechanochemical Control of Stem Cell Biology in Development and Disease: Experimental and Theoretical Models

Whether a stem cell remains or egresses away from its physiological niche is a function of mechanical and soluble factors in a time-dependent manner, which implicates a `memory\u27 of prior mechanochemical conditioning. Virtually every organ in the body contains resident stem or progenitor cells that contribute to organ homeostasis or repair. The wound healing process in higher vertebrate animals is spatiotemporally complex and usually leads to scarring. Limitations for the use of stem cells as regenerative therapy include the lack of expansion capabilities in vitro as well as materials issues that complicate traditional biochemical protocols. A minimal `scar in a dish\u27 model is developed to clarify the kinetics of tension-sensitive proteins in mesenchymal stem cells (MSCs), which possess plasticity to mechanochemical changes of the microenvironment that are typical of scars. The organization and expression of such proteins implicates transcription factors that ultimately steer cell fate. In contrast to classic mechano-transducers of matrix mechanics such as actin assembly-dependent serum response factor (SRF) signaling, a novel mechano-repressive role of NKX2.5 is implicated in maintaining intracellular tension in long-term stem cell cultures on stiff matrices via nucleo-cytoplasmic shuttling — ultimately setting up a \u27mechanical memory\u27. Core gene circuits with known roles in stem cell mechanobiology are modeled based on the \u27use it or lose it\u27 concept: tension inhibits turnover of structural proteins such as extracellular collagens, cytoskeletal myosins and nucleoskeletal lamins. This theoretical approach is tested in a variety of processes in vitro and in vivo that involve forces including cardiac development, osteogenic commitment of MSCs, and fibrosis therapy. With the sophistication of the science and technology of biomaterials relevant to stem cell biology and medicine, matrix mechanics can thus be rigorously combined with biochemical instructions in order to maximize therapeutic utility of stem cells

Regulation of Vg1 biogenesis during mesendoderm induction

The TGF-beta signals Vg1 and Nodal form heterodimers to induce the vertebrate mesendoderm. The Vg1 proprotein is a monomer retained in the endoplasmic reticulum (ER) and is processed and secreted upon heterodimerization with Nodal. Here we investigate the mechanisms underlying Vg1 retention, processing, secretion and signaling in zebrafish. First, using a newly devised Synthetic Processing (SynPro) system, we find that Vg1 can be processed by intra- or extracellular proteases. Second, Vg1 can be processed without Nodal but requires Nodal for secretion and signaling. Third, Vg1-Nodal signaling activity requires Vg1 processing, whereas Nodal can remain unprocessed. Fourth, Vg1 employs exposed cysteines, glycosylated asparagines, and BiP chaperone-binding motifs for monomer retention in the ER. Our results establish SynPro as a new in vivo processing system and define molecular mechanisms and motifs that facilitate the generation of active Vg1-Nodal heterodimers. These observations suggest two strategies for rapid mesendoderm induction: chaperone-binding motifs help store Vg1 as an inactive but ready-to-heterodimerize monomer in the ER, and the flexibility of Vg1 processing location allows efficient generation of active heterodimers both intra- and extracellularly

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors.

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage

A mathematical model of mechanotransduction reveals how mechanical memory regulates mesenchymal stem cell fate decisions

Abstract Background Mechanical and biophysical properties of the cellular microenvironment regulate cell fate decisions. Mesenchymal stem cell (MSC) fate is influenced by past mechanical dosing (memory), but the mechanisms underlying this process have not yet been well defined. We have yet to understand how memory affects specific cell fate decisions, such as the differentiation of MSCs into neurons, adipocytes, myocytes, and osteoblasts. Results We study a minimal gene regulatory network permissive of multi-lineage MSC differentiation into four cell fates. We present a continuous model that is able to describe the cell fate transitions that occur during differentiation, and analyze its dynamics with tools from multistability, bifurcation, and cell fate landscape analysis, and via stochastic simulation. Whereas experimentally, memory has only been observed during osteogenic differentiation, this model predicts that memory regions can exist for each of the four MSC-derived cell lineages. We can predict the substrate stiffness ranges over which memory drives differentiation; these are directly testable in an experimental setting. Furthermore, we quantitatively predict how substrate stiffness and culture duration co-regulate the fate of a stem cell, and we find that the feedbacks from the differentiating MSC onto its substrate are critical to preserve mechanical memory. Strikingly, we show that re-seeding MSCs onto a sufficiently soft substrate increases the number of cell fates accessible. Conclusions Control of MSC differentiation is crucial for the success of much-lauded regenerative therapies based on MSCs. We have predicted new memory regions that will directly impact this control, and have quantified the size of the memory region for osteoblasts, as well as the co-regulatory effects on cell fates of substrate stiffness and culture duration. Taken together, these results can be used to develop novel strategies to better control the fates of MSCs in vitro and following transplantation

Letter from S. Dingal, Associate Editor at 'The Day,' to Mr. Baruch Drazin, Chairman of the Board of Directors of the Hightstown Colony, July 31, 1936

Jersey Homesteads (later Roosevelt) was established in the 1930s as an agro-industrial cooperative community. It was established specifically for urban Jewish garment workers, many of whom had emigrated from Europe. S. Dingal, the Associate Editor of the national Jewish newspaper, 'The Day,' wrote a letter to Baruch [Boris] Drazin, the Chairman of the Board of Directors of the Hightstown Colony, apologizing for not being able to attend the opening ceremony for the factory on the following Sunday. Dingal sent his wishes for a successful opening of the factory and believed that the factory would just be the beginning of cooperative factories in America

Antimicrobial efficacy testing of antibiotic-containing biodegradable nanopolymers against biofilm and planktonic cells.

Cystic fibrosis and rampant urogenital infections, caused by increasingly resistant microbial biofilms, call for more creative anti-infective systems. This study investigated the in vitro efficacy of levofloxacin (LEV)–loaded poly(D,L-lactide-co-glycolide) (PLGA) and poly-ε-caprolactone (PCL) nanopolymers against optimally-grown biofilms of Escherichia coli K12 W3110 and Pseudomonas aeruginosa PA01. High-throughput biofilm production and antimicrobial susceptibility testing were conducted in the Calgary Biofilm Device (CBD). Rich Luria-Bertani medium provided maximal accumulation of biofilm biomass, with optimum times (24 and 48 h, respectively) and temperatures (30 C and 21 C, respectively) found under dynamic culture conditions. For both pathogens, minimum inhibitory concentrations (MIC) of 2.8 µg/mL and 16.5 µg/mL total LEV load were found for LEV-PLGA and LEV-PCL, respectively. Minimum biofilm eradication concentrations (MBECs) improved at least 2-fold with increase in exposure time (48 hours) achieving LEV-PLGA MBECs of 1.4 and 0.2 µg/mL and LEV-PCL MBECs of 16.5 and 8.2 µg/mL for E. coli and P. aeruginosa, respectively. With efficient use of drug-encapsulated nanopolymers, bactericidal dosages are sufficiently lowered and dosing intervals can be extended due to the sustained drug release feature afforded by these efficacious nanocarriers.Bachelor of Engineering (Chemical and Biomolecular Engineering

Modeling Developmental Signaling Proteins Using Alphafold2

Vertebrate animal development is governed by the signaling proteins that pattern fields of embryonic cells and direct their intended tissue fate. Vg1 and Nodal are two such signaling proteins that induce the formation of mesoderm and endoderm tissues (e.g., muscle, bone, blood tissues). Vg1 and Nodal are members of the transforming growth factor-beta superfamily of proteins that have no existing crystal structures yet. We utilized Alphafold2, a deep learning algorithm, to computationally predict (1) the monomeric structures of Vg1, Nodal, their receptors, agonists and antagonists; and (2) the heteromeric structures that will indicate amino acid residues potentially involved in their interactions and downstream signaling. We will show several Alphafold2 models of the multi-protein complex of Vg1-Nodal bound to their receptors, which inspired multiple exciting hypotheses that we now plan to experimentally validate.Texas Advanced Computing Center (TACC