Junaid Afzal

Spatial control of adult stem cell fate using nanotopographic cues

Adult stem cells hold great promise as a source of diverse terminally differentiated cell types for tissue engineering applications. However, due to the complexity of chemical and mechanical cues specifying differentiation outcomes, development of arbitrarily complex geometric and structural arrangements of cells, adopting multiple fates from the same initial stem cell population, has been difficult. Here, we show that the topography of the cell adhesion substratum can be an instructive cue to adult stem cells and topographical variations can strongly bias the differentiation outcome of the cells towards adipocyte or osteocyte fates. Switches in cell fate decision from adipogenic to osteogenic lineages were accompanied by changes in cytoskeletal stiffness, spanning a considerable range in the cell softness/rigidity spectrum. Our findings suggest that human mesenchymal stem cells (hMSC) can respond to the varying density of nanotopographical cues by regulating their internal cytoskeletal network and use these mechanical changes to guide them toward making cell fate decisions. We used this finding to design a complex two-dimensional pattern of co-localized cells preferentially adopting two alternative fates, thus paving the road for designing and building more complex tissue constructs with diverse biomedical applications.

Matrix Rigidity Controls Endothelial Differentiation and Morphogenesis of Cardiac Precursors

Culturing cardiac precursors on a surface with the rigidity of heart tissue increases the yield of endothelial cells. The Heart of the Matrix Cardiosphere-derived cells (CDCs) are adult stem cells with the potential to differentiate into endothelial cells and cardiomyocytes, which are the major cell types of the heart. Their potential to induce cardiac regeneration after myocardial infarct is currently being clinically tested. Kshitiz et al. showed that CDCs cultured on a substrate with a rigidity matching that of normal myocardium yielded higher proportions of cells with the adhesion molecule CD31 (a marker of differentiated endothelial cells) than did those cultured on less or more rigid substrata. CDCs cultured on substratum equivalent in rigidity to the myocardium developed into organized cellular networks reminiscent of blood vessels and appeared to integrate more efficiently into the vasculature of ischemic rat myocardium. The process of sensing substratum rigidity occurred throughout the in vitro culture period, and the signaling pathway involved required the guanosine triphosphatase (GTPase)–activating protein p190RhoGAP acting through various downstream effectors, including the GTPase RhoA. These results could potentially increase the efficacy of regenerative therapies that use CDCs to repair hearts after myocardial infarction. Tissue development and regeneration involve tightly coordinated and integrated processes: selective proliferation of resident stem and precursor cells, differentiation into target somatic cell type, and spatial morphological organization. The role of the mechanical environment in the coordination of these processes is poorly understood. We show that multipotent cells derived from native cardiac tissue continually monitored cell substratum rigidity and showed enhanced proliferation, endothelial differentiation, and morphogenesis when the cell substratum rigidity closely matched that of myocardium. Mechanoregulation of these diverse processes required p190RhoGAP, a guanosine triphosphatase–activating protein for RhoA, acting through RhoA-dependent and -independent mechanisms. Natural or induced decreases in the abundance of p190RhoGAP triggered a series of developmental events by coupling cell-cell and cell-substratum interactions to genetic circuits controlling differentiation.

Concise Review: Mechanotransduction via p190RhoGAP Regulates a Switch Between Cardiomyogenic and Endothelial Lineages in Adult Cardiac Progenitors

Mechanical cues can have pleiotropic influence on stem cell shape, proliferation, differentiation, and morphogenesis, and are increasingly realized to play an instructive role in regeneration and maintenance of tissue structure and functions. To explore the putative effects of mechanical cues in regeneration of the cardiac tissue, we investigated therapeutically important cardiosphere‐derived cells (CDCs), a heterogeneous patient‐ or animal‐specific cell population containing c‐Kit+ multipotent stem cells. We showed that mechanical cues can instruct c‐Kit+ cell differentiation along two lineages with corresponding morphogenic changes, while also serving to amplify the initial c‐Kit+ subpopulation. In particular, mechanical cues mimicking the structure of myocardial extracellular matrix specify cardiomyogenic fate, while cues mimicking myocardium rigidity specify endothelial fates. Furthermore, we found that these cues dynamically regulate the same molecular species, p190RhoGAP, which then acts through both RhoA‐dependent and independent mechanisms. Thus, differential regulation of p190RhoGAP molecule by either mechanical inputs or genetic manipulation can determine lineage type specification. Since human CDCs are already in phase II clinical trials, the potential therapeutic use of mechanical or genetic manipulation of the cell fate could enhance effectiveness of these progenitor cells in cardiac repair, and shed new light on differentiation mechanisms in cardiac and other tissues. Stem Cells 2014;32:1999–2007

A nanotopography approach for studying the structure-function relationships of cells and tissues

Most cells in the body secrete, or are in intimate contact with extracellular matrix (ECM), which provides structure to tissues and regulates various cellular phenotypes. Cells are well known to respond to biochemical signals from the ECM, but recent evidence has highlighted the mechanical properties of the matrix, including matrix elasticity and nanotopography, as fundamental instructive cues regulating signal transduction pathways and gene transcription. Recent observations also highlight the importance of matrix nanotopography as a regulator of cellular functions, but lack of facile experimental platforms has resulted in a continued negligence of this important microenvironmental cue in tissue culture experimentation. In this review, we present our opinion on the importance of nanotopography as a biological cue, contexts in which it plays a primary role influencing cell behavior, and detail advanced techniques to incorporate nanotopography into the design of experiments, or in cell culture environments. In addition, we highlight signal transduction pathways that are involved in conveying the extracellular matrix nanotopography information within the cells to influence cell behavior.

Cellular Bioenergetics Is an Important Determinant of the Molecular Imaging Signal Derived From Luciferase and the Sodium-Iodide Symporter

Rationale: Molecular imaging is useful for longitudinal assessment of engraftment. However, it is not known which factors, other than cell number, can influence the molecular imaging signal obtained from reporter genes. Objective: The effects of cell dissociation/suspension on cellular bioenergetics and the signal obtained by firefly luciferase and human sodium-iodide symporter labeling of cardiosphere-derived cells were investigated. Methods and Results: 18Fluorodeoxyglucose uptake, ATP levels, 99mTc-pertechnetate uptake, and bioluminescence were measured in vitro in adherent and suspended cardiosphere-derived cells. In vivo dual-isotope single-photon emission computed tomography/computed tomography imaging or bioluminescence imaging (BLI) was performed 1 hour and 24 hours after cardiosphere-derived cell transplantation. Single-photon emission computed tomography quantification was performed using a phantom for signal calibration. Cell loss between 1 hour and 24 hours after transplantation was quantified by quantitative polymerase chain reaction and ex vivo luciferase assay. Cell dissociation followed by suspension for 1 hour resulted in decreased glucose uptake, cellular ATP, 99mTc uptake, and BLI signal by 82%, 43%, 42%, and 44%, respectively, compared with adherent cells, in vitro. In vivo 99mTc uptake was significantly lower at 1 hour compared with 24 hours after cell transplantation in the noninfarct (P<0.001; n=3) and infarct (P<0.001; n=4) models, despite significant cell loss during this period. The in vivo BLI signal was significantly higher at 1 hour than at 24 hours (P<0.01), with the BLI signal being higher when cardiosphere-derived cells were suspended in glucose-containing medium compared with saline (PBS). Conclusions: Adhesion is an important determinant of cellular bioenergetics, 99mTc-pertechnetate uptake, and BLI signal. BLI and sodium-iodide symporter imaging may be useful for in vivo optimization of bioenergetics in transplanted cells.

Control of the interface between heterotypic cell populations reveals the mechanism of intercellular transfer of signaling proteins

Direct intercellular transfer of cellular components is a recently described general mechanism of cell–cell communication. It is a more non-specific mode of intercellular communication that is not actively controlled by the participating cells. Though membrane bound proteins and small non-protein cytosolic components have been shown to be transferred between cells, the possibility of transfer of cytosolic proteins has not been clearly established, and its mechanism remains unexplained. Using a cell–cell pair of metastatic melanoma and endothelial cells, known to interact at various stages during cancer progression, we show that cytosolic proteins can indeed be transferred between heterotypic cells. Using precise relative cell patterning we provide evidence that this transfer depends on extent of the interface between heterotypic cell populations. This result is further supported by a mathematical model capturing various experimental conditions. We further demonstrate that cytosolic protein transfer can have important functional consequences for the tumor–stroma interactions, e.g., in heterotypic transfer of constitutively activated BRAF, a common melanoma associated mutation, leading to an enhanced activation of the downstream MAPK pathway. Our results suggest that cytosolic protein transfer can have important consequences for regulation of processes involving physical co-location of heterotypic cell types, particularly in invasive cancer growth.

Cardiosphere-Derived Cells Demonstrate Metabolic Flexibility That Is Influenced by Adhesion Status

Visual Abstract

Mechanics of Microenvironment as Instructive Cues Guiding Stem Cell Behavior

Stem cells reside in a complex milieu during development, or in adult tissues, as well as in culture conditions. Their decision to differentiate, self-renew, or migrate is a result of an integrated response to extracellular stimuli, which are chemical, physical, and mechanical in nature. In recent years, research has shown that the mechanical properties of the microenvironment can regulate a variety of stem cell phenotypes by activating intracellular signal transduction leading to transcription. Many of these signaling pathways are primarily involved in mechanotransduction, suggesting that mechanical cues, particularly the rigidity and topographical architecture of the extracellular matrix directly regulate stem cell behavior. Novel bioengineering tools have made it possible for the first time to systematically and quantifiably understand the role of mechanical cues in stem cell biology. However, it is necessary to investigate activation of mechanotransduction in the context of other signals to which cells respond. How cells integrate complex presentation of signals, including mechanical cues, to formulate a decision will increase our understanding of fundamental stem cell biology, as well as inform future therapeutic applications in regenerative medicine.