Scaffold dimensionality and confinement determine single cell morphology and migration

This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics

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

Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics

In response to αβ1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require αβ1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of αβ1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL–PIX–PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK

Mechanosensing By The Nuclear Lamina: From Embryonic Development To Aging

‘Nuclear mechanosensing’ encompasses a wide range of biophysical pathways that are emerging as key processes in the regulation of cell function and fate. Many of these mechanisms involve the main structural protein of the nucleus, lamin-A, which is abundant in stiff and mechanically stressed tissues such as striated muscle, but is comparatively low in soft tissues such as the brain. Lamin-A’s increase with tissue stiffness correlates strongly with elevated levels of collagen-I fibers in the extracellular matrix (ECM), but mechanisms and functional consequences of any matrix-nucleus interplay remain unclear. Here, in the first set of studies, we show that lamin-A and collagen-I exhibit tightly coupled mechano-sensitivity in the first functional vertebrate organ, the beating embryonic heart, following a mechanism for tension-suppressed turnover that confers mechano-protection against DNA damage. Lamin-A and collagen-I increase together as the heart stiffens daily in embryogenesis, but their levels are found here to be modulated within 1-2 hours by rapid and reversible perturbations of actomyosin contractility or ECM mechanics. In both intact hearts and in isolated cardiomyocytes, suppression of lamin-A – combined with high contractile stress – results in i) increased nuclear envelope rupture, ii) cytoplasmic mis-localization of DNA repair factors, and iii) accumulation of DNA damage, which ultimately causes arrythmia. Embryonic cardiomyocytes on stiff collagen-coated gels show increased lamin-A levels compared to those on soft gels, suggesting a cell-intrinsic protective mechanism against DNA damage. Interphase phosphorylation of lamin-A emerges as a key posttranslational modification that gives rise to such mechano-sensitivity, as phosphorylation and subsequent degradation of lamin-A are suppressed with myosin-II-dependent cell spreading. This mechanism of tension-suppressed turnover is further examined in a second set of studies, which focuses on the aging-associated lamin-A mutant, ‘progerin’. Using a novel mass spectrometry-based workflow, we find that progerin phosphorylation in patient iPS-derived cells is lower and less mechanosensitive compared to normal lamin-A and C, suggesting that a loss in the nucleus’ ability to dynamically remodel in response to stress could contribute to genome instability and aging. Mechanosensing by lamin-A is thus critical not only in embryonic development, but also in disease and aging of mature tissues

Actin dynamics regulation by TTC7A/PI4KIIIα limits DNA damage and cell death under confinement

Background: The actin cytoskeleton has a crucial role in the maintenance of the immune homeostasis by controlling various cellular processes, including cell migration. Mutations in TTC7A have been described as the cause of a primary immunodeficiency associated to different degrees of gut involvement and alterations in the actin cytoskeleton dynamics. Objectives: This study investigates the impact of TTC7A deficiency in immune homeostasis. In particular, the role of the TTC7A/phosphatidylinositol 4 kinase type III α pathway in the control of leukocyte migration and actin dynamics. Methods: Microfabricated devices were leveraged to study cell migration and actin dynamics of murine and patient-derived leukocytes under confinement at the single-cell level. Results: We show that TTC7A-deficient lymphocytes exhibit an altered cell migration and reduced capacity to deform through narrow gaps. Mechanistically, TTC7A-deficient phenotype resulted from impaired phosphoinositide signaling, leading to the downregulation of the phosphoinositide 3-kinase/AKT/RHOA regulatory axis and imbalanced actin cytoskeleton dynamics. TTC7A-associated phenotype resulted in impaired cell motility, accumulation of DNA damage, and increased cell death in dense 3-dimensional gels in the presence of chemokines. Conclusions: These results highlight a novel role of TTC7A as a critical regulator of lymphocyte migration. Impairment of this cellular function is likely to contribute to the pathophysiology underlying progressive immunodeficiency in patients.</p

Blueprint for an intestinal villus: Species‐specific assembly required

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144650/1/wdev317_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144650/2/wdev317.pd

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

In vitro microphysiological system for modeling vascular disease

In vitro microphysiological system utilizes engineered tissue constructs from human cells to model functional activity of human tissues or organs in both healthy and diseased state, thereby providing a more accurate drug screening than animal models prior to clinical trials. One essential component of an in vitro microphysiological system is a tissue engineered blood vessel (TEBV) that can accurately recapitulate the functional vasculature in vivo. This thesis first explores two most important considerations to a successful TEBV generation, the cell source and the fabrication method. To engineer a vascular tissue construct, an ideal cell source should demonstrate high availability and accurate vessel functionality. Mesenchymal stem cells (MSC) were explored due to their high availability, proliferation capacity, and capability to deposit adequate extracellular matrix (ECM) for cell sheet formation. Vascular smooth muscle cells (SMC) are the cell components that comprise the medial layer of native blood vessel, and thus optimal for demonstrating equivalent biological functionality. However, SMC are much harder to acquire through biopsy, and they have limited proliferative capacity and quick senescence. Therefore, an alternative cell source for SMC was obtained through direct reprogramming approach involving the induced overexpression of myocardin in more readily available human cell sources. The resulting reprogrammed SMC demonstrated close resemblance to the native SMC in terms of its phenotype, related gene and protein expression levels, and contractile function. Two different fabrication methods, nanopatterned cell sheets and dense collagen hydrogel, were explored to engineer a 1 mm inner diameter blood vessel. The fabricated TEBVs were then compared to that of the native blood vessel and each other in terms of its structure, mechanical properties, and vasoactive function in response to stimuli. After selecting the most optimal cell source and fabrication method for developing a human cell-based TEBV for in vitro microphysiological system, the second part of this thesis assesses the capability of the designed TEBV to model a vascular disease for drug screening purposes. Marfan syndrome was selected as a model vascular disease due to its previous history of contradictory results from the animal models and human clinical trials using losartan, an angiotensin II receptor blocker, in terms of preventing aortic root dilation. TEBV fabricated using reprogrammed SMC from Marfan syndrome patient sample and dense collagen hydrogel showed reduced fibrillin deposition, increased vessel diameter and thickness, and reduced vasoconstriction levels when compared to the wild type TEBV, which is consistent with that observed in native vessels of Marfan syndrome patients. Losartan improved the function of Marfan syndrome TEBV, but still at reduced level when compared to that of the wild type. SB203580, a selective inhibitor of p53 MAPK that has been shown to be a better drug candidate than losartan in recent cell-based studies, showed improved TEBV function comparable to that of the wild type. In overall, this thesis presents a successful development of a highly robust, patient-specific in vitro vascular model. An accurate recapitulation of a drug-induced physiological response in humans can speed up the drug screening process with higher efficiency, and this will eventually increase the chances of successful treatment for patients

Nicotinamide benefits both mothers and pups in two contrasting mouse models of preeclampsia

Preeclampsia (PE), high blood pressure and protein in the urine in the last third of pregnancy, complicates about 1 in 20 human pregnancies, and it is one of the leading causes of pregnancy-related maternal deaths. The only definitive treatment, induced delivery, invariably results in premature babies. Blood pressure-lowering drugs help, but results in preventing preterm delivery and correcting the fetal growth restriction (FGR) that also occurs in PE have been disappointing. Here we show that feeding high doses of nicotinamide, a vitamin, improves the maternal condition, prolongs pregnancies, and prevents FGR in mice having PE-like conditions due to two contrasting causes. Because nicotinamide benefits both mothers and pups, it merits evaluation for preventing or treating PE in humans