Yue Shao

Integrated micro/nanoengineered functional biomaterials for cell mechanics and mechanobiology: a materials perspective.

The rapid development of micro/nanoengineered functional biomaterials in the last two decades has empowered materials scientists and bioengineers to precisely control different aspects of the in vitro cell microenvironment. Following a philosophy of reductionism, many studies using synthetic functional biomaterials have revealed instructive roles of individual extracellular biophysical and biochemical cues in regulating cellular behaviors. Development of integrated micro/nanoengineered functional biomaterials to study complex and emergent biological phenomena has also thrived rapidly in recent years, revealing adaptive and integrated cellular behaviors closely relevant to human physiological and pathological conditions. Working at the interface between materials science and engineering, biology, and medicine, we are now at the beginning of a great exploration using micro/nanoengineered functional biomaterials for both fundamental biology study and clinical and biomedical applications such as regenerative medicine and drug screening. In this review, an overview of state of the art micro/nanoengineered functional biomaterials that can control precisely individual aspects of cell-microenvironment interactions is presented and they are highlighted them as well-controlled platforms for mechanistic studies of mechano-sensitive and -responsive cellular behaviors and integrative biology research. The recent exciting trend where micro/nanoengineered biomaterials are integrated into miniaturized biological and biomimetic systems for dynamic multiparametric microenvironmental control of emergent and integrated cellular behaviors is also discussed. The impact of integrated micro/nanoengineered functional biomaterials for future in vitro studies of regenerative medicine, cell biology, as well as human development and disease models are discussed.

On human pluripotent stem cell control: The rise of 3D bioengineering and mechanobiology.

Human pluripotent stem cells (hPSCs) provide promising resources for regenerating tissues and organs and modeling development and diseases in vitro. To fulfill their promise, the fate, function, and organization of hPSCs need to be precisely regulated in a three-dimensional (3D) environment to mimic cellular structures and functions of native tissues and organs. In the past decade, innovations in 3D culture systems with functional biomaterials have enabled efficient and versatile control of hPSC fate at the cellular level. However, we are just at the beginning of bringing hPSC-based regeneration and development and disease modeling to the tissue and organ levels. In this review, we summarize existing bioengineered culture platforms for controlling hPSC fate and function by regulating inductive mechanical and biochemical cues coexisting in the synthetic cell microenvironment. We highlight recent excitements in developing 3D hPSC-based in vitro tissue and organ models with in vivo-like cellular structures, interactions, and functions. We further discuss an emerging multifaceted mechanotransductive signaling network--with transcriptional coactivators YAP and TAZ at the center stage--that regulate fates and behaviors of mammalian cells, including hPSCs. Future development of 3D biomaterial systems should incorporate dynamically modulated mechanical and chemical properties targeting specific intracellular signaling events leading to desirable hPSC fate patterning and functional tissue formation in 3D.

Uniaxial cell stretching device for live-cell imaging of mechanosensitive cellular functions

External mechanical stretch plays an important role in regulating cellular behaviors through intracellular mechanosensitive and mechanotransductive machineries such as the F-actin cytoskeleton (CSK) structures and focal adhesions (FAs) anchoring the F-actin CSK to the extracellular environment. Studying the mechanoresponsive behaviors of the F-actin CSK and FAs in response to cell stretch has great importance for further understanding mechanotransduction and mechanobiology. In this work, we developed a novel cell stretching device combining dynamic directional cell stretch with in situ subcellular live-cell imaging. Using a cam and follower mechanism and applying a standard mathematical model for cam design, we generated different dynamic stretch outputs. By examining stretch-mediated FA dynamics under step-function static stretch and the realignment of cell morphology and the F-actin CSK under cyclic stretch, we demonstrated successful applications of our cell stretching device for mechanobiology studies where external stretch plays an important role in regulating subcellular molecular dynamics and cellular phenotypes.

Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells

Summary We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.

A pluripotent stem cell-based model for post-implantation human amniotic sac development

Development of the asymmetric amniotic sac—with the embryonic disc and amniotic ectoderm occupying opposite poles—is a vital milestone during human embryo implantation. Although essential to embryogenesis and pregnancy, amniotic sac development in humans remains poorly understood. Here, we report a human pluripotent stem cell (hPSC)-based model, termed the post-implantation amniotic sac embryoid (PASE), that recapitulates multiple post-implantation embryogenic events centered around amniotic sac development. Without maternal or extraembryonic tissues, the PASE self-organizes into an epithelial cyst with an asymmetric amniotic ectoderm-epiblast pattern that resembles the human amniotic sac. Upon further development, the PASE initiates a process that resembles posterior primitive streak development in a SNAI1-dependent manner. Furthermore, we observe asymmetric BMP-SMAD signaling concurrent with PASE development, and establish that BMP-SMAD activation/inhibition modulates stable PASE development. This study reveals a previously unrecognized fate potential of human pluripotent stem cells and provides a platform for advancing human embryology.Early in human embryonic development, it is unclear how amniotic sac formation is regulated. Here, the authors use a human pluripotent stem cell-based model, termed the post-implantation amniotic sac embryoid, to recapitulate early embryogenic events of human amniotic sac development.

Global architecture of the F-actin cytoskeleton regulates cell shape-dependent endothelial mechanotransduction

Uniaxial stretch is an important biophysical regulator of cell morphology (or shape) and functions of vascular endothelial cells (ECs). However, it is unclear whether and how cell shape can independently regulate EC mechanotransductive properties under uniaxial stretch. Herein, utilizing a novel uniaxial cell-stretching device integrated with micropost force sensors, we reported the first experimental evidence showing cell shape-dependent EC mechanotransduction via cytoskeleton (CSK) contractile forces in response to uniaxial stretch. Combining experiments and theoretical modeling from first principles, we showed that it was the global architecture of the F-actin CSK that instructed the cell shape-dependent EC mechanotransductive process. Furthermore, a cell shape-dependent nature was relayed in EC mechanotransduction via dynamic focal adhesion (FA) assembly. Our results suggested a novel mechanotransductive process in ECs wherein the global architecture of the F-actin CSK, governed by cell shape, controls mechanotransduction via CSK contractile forces and force-dependent FA assembly under uniaxial stretch.

Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis.

Mechanical homeostasis - a fundamental process by which cells maintain stable states under environmental perturbations - is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs)1-5. Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths via either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modeling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviors in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight previously underappreciated physical nature of the mechanical homeostasis of cells.

Angiogenesis in Liquid Tumors: An In Vitro Assay for Leukemic Cell Induced Bone Marrow Angiogenesis

The critical role of angiogenesis for solid tumor growth and metastatic spread has been well established. In contrast, even though increased vascularity has been commonly observed in bone marrows of patients with hematological malignancies (liquid tumors), the pathophysiology of leukemia-induced angiogenesis in the bone marrow remains elusive. This paper demonstrates the usage of a microengineered 3D biomimetic model to study leukemic-cell-induced bone marrow angiogenesis. Rational design of the 3D angiogenesis chip incorporating endothelial cells (ECs), leukemic cells, and bone marrow stromal fibroblasts provide an efficient biomimetic means to promote and visualize early angiogenic processes. Morphological features of angiogenesis induced by three different leukemic cell lines (U937, HL60, and K562) are investigated and compared. Quantitative measurements of angiogenic factors secreted from monocultures and cocultures of leukemic cells with bone marrow stromal fibroblasts suggest a synergistic relationship between ECs, leukemic cells, and bone marrow stromal fibroblasts for angiogenic induction, and also confirm the necessity of conducting functional angiogenic assays in proper 3D biomimetic cell culture systems like the one developed in this work.

A Miniaturized Hemoretractometer for Blood Clot Retraction Testing

Blood coagulation is a critical hemostatic process that must be properly regulated to maintain a delicate balance between bleeding and clotting. Disorders of blood coagulation can expose patients to the risk of either bleeding disorders or thrombotic diseases. Coagulation diagnostics using whole blood is very promising for assessing the complexity of the coagulation system and for global measurements of hemostasis. Despite the clinic values that existing whole blood coagulation tests have demonstrated, these systems have significant limitations that diminish their potential for point-of-care applications. Here, recent advancements in device miniaturization using functional soft materials are leveraged to develop a miniaturized clot retraction force assay device termed mHemoRetractoMeter (mHRM). The mHRM is capable of precise measurements of dynamic clot retraction forces in real time using minute amounts of whole blood. To further demonstrate the clinical utility of the mHRM, systematic studies are conducted using the mHRM to examine the effects of assay temperature, treatments of clotting agents, and pro- and anti-coagulant drugs on clot retraction force developments of whole blood samples. The mHRMs low fabrication cost, small size, and consumption of only minute amounts of blood samples make the technology promising as a point-of-care tool for future coagulation monitoring.

Atomic force microscopy indentation and inverse analysis for non-linear viscoelastic identification of breast cancer cells

Breast cancer cells (MCF-7 and MCF-10A) are studied through indentation with spherical borosilicate glass particles in atomic force microscopy (AFM) contact mode in fluid. Their mechanical properties are obtained by analyzing the recorded reaction force-time response. The analysis is based on comparing experimental data with predictions from finite element (FE) simulation. Here, FE modeling is employed to simulate the AFM indentation experiment which is neither a displacement nor a force controlled test. This approach is expected to overcome many underlying problems of the widely used models such as Hertz contact model due to its capability to capture the contact behaviors between the spherical indentor and the cell, account for cell geometry, and incorporate with large strain theory. In this work, a non-linear viscoelastic (NLV) model in which the viscoelastic part is described by Prony series terms is used for the constitutive model of the cells. The time-dependent material parameters are extracted through an inverse analysis with the use of a surrogate model based on a Kriging estimator. The purpose is to automatically extract the NLV properties of the cells with a more efficient process compared to the iterative inverse technique that has been mostly applied in the literature. The method also allows the use of FE modeling in the analysis of a large amount of experimental data. The NLV parameters are compared between MCF-7 and MCF-10A and MCF-10A treated and untreated with the drug Cytochalasin D to examine the possibility of using relaxation properties as biomarkers for distinguishing these types of breast cancer cells. The comparisons indicate that malignant cells (MCF-7) are softer and exhibit more relaxation than benign cells (MCF-10A). Disrupting the cytoskeleton using the drug Cytochalasin D also results in a larger amount of relaxation in the cells response. In addition, relaxation properties indicate larger differences as compared to the elastic moduli like instantaneous shear modulus. These results may be useful for disease diagnosing purposes.