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Trophoblast stem cells (TSCs) are a heterogeneous cell population despite the presence of fibroblast growth factor (FGF) and transforming growth factor β (TGFB) as key growth factors in standard culture conditions. To understand what other signaling cascades control the stem cell state of mouse TSCs, we performed a kinase inhibitor screen and identified several novel pathways that cause TSC differentiation. Surprisingly, inhibition of phosphoinositide-3-kinase (PI3K) signaling increased the mRNA and protein expression of stem cell markers instead, and resulted in a tighter epithelial colony morphology and fewer differentiated cells. PI3K inhibition could not substitute for FGF or TGFB and did not affect phosphorylation of extracellular signal-regulated kinase, and thus acts independently of these pathways. Upon removal of PI3K inhibition, TSC transcription factor levels reverted to normal TSC levels, indicating that murine TSCs can reversibly switch between these two states. In summary, PI3K inhibition reduces the heterogeneity and seemingly heightens the stem cell state of TSCs as indicated by the simultaneous upregulation of multiple key marker genes and cell morphology. Stem Cells 2019;37:1307–1318

Expression of E-cadherin is used to monitor the epithelial phenotype, and its loss is suggestive of epithelial-mesenchymal transition (EMT). EMT triggers tumor metastasis. Exit from EMT is marked by increased E-cadherin expression and is considered necessary for tumor growth at sites of metastasis; however, the mechanisms associated with exit from EMT are poorly understood. Herein are analyzed 185 prostate cancer metastases, with significantly higher E-cadherin expression in bone than in lymph node and soft tissue metastases. To determine the molecular mechanisms of regulation of E-cadherin expression, three stable isogenic cell lines from DU145 were derived that differ in structure, migration, and colony formation on soft agar and Matrigel. When injected into mouse tibia, the epithelial subline grows most aggressively, whereas the mesenchymal subline does not grow. In cultured cells, ZEB1 and Src family kinases decrease E-cadherin expression. In contrast, in tibial xenografts, E-cadherin RNA levels increase eight- to 10-fold despite persistent ZEB1 expression, and in all ZEB1-positive metastases (10 of 120), ZEB1 and E-cadherin proteins were co-expressed. These data suggest that transcriptional regulation of E-cadherin differs in cultured cells versus xenografts, which more faithfully reflect E-cadherin regulation in cancers in human beings. Furthermore, the aggressive nature of xenografts positive for E-cadherin and the frequency of metastases positive for E-cadherin suggest that high E-cadherin expression in metastatic prostate cancer is associated with aggressive tumor growth.

Adipose tissue is an attractive source of multipotent adult stem cells due to its wide-spread availability, accessibility, and ease of harvest. Adipose-derived stem cells (ASCs), the adherent stromal cell population present within adipose tissue, are easily expanded in culture, able to differentiate along multiple cell-lineage pathways, and have been shown to provide therapeutic benefit in models of injury and disease through immunomodulation, structural integation, and/or trophic support. Recent developments in the characterization of ASCs, specifically their isolation, gene and protein expression, differentiation, and expansion, are reviewed in this article.

Skeletal muscle vasculature undergoes arteriogenesis to restore tissue perfusion and function following loss of blood flow. This process has been shown to occur in large vessels following ischemia, although recent studies suggest this may occur in the microcirculation as well. We tested the hypothesis that ischemia induces microvascular remodeling in the skeletal muscle microcirculation on the scale of capillary to sub-35 mum diameter arterioles.Ligations of a feeding arteriole to the caudal-half of the spinotrapezius muscle were performed on C57BL/6 mice. At 5 days, microvascular remodeling responses were quantified using intravital and whole-mount confocal microscopy. Immunohistochemistry was performed to visualize vessels, incorporated leukocytes, and regions of hypoxia.Ischemic tissue underwent localized microvascular remodeling characteristic of arteriogenesis, including pronounced vessel tortuosity. In patent microvessels (diameters 15-35 microm), we observed increases in vascular density (38%), branching (90%) and collateral development (36.5%). The formation of new arterioles (diameters 6-35 microm) increased by 24.3%, while chronic hypoxia was absent from all tissues.Ischemic injury induces arteriogenesis in skeletal muscle microcirculation. Furthermore, this surgical model enables en face analysis of microcirculatory adaptations with single-cell resolution and can provide investigators with morphometric data on a microscale that is difficult to achieve using other models.

Leukocyte trafficking through the microcirculation and into tissues is central in angiogenesis, inflammation, and the immune response. Although the literature is rich with mechanistic detail describing molecular mediators of these processes, integration of signaling events and cell behaviors within a unified spatial and temporal framework at the multi-cell tissue-level is needed to achieve a fuller understanding. We have developed a novel computational framework that combines agent-based modeling (ABM) with a network flow analysis to study monocyte homing. A microvascular network architecture derived from mouse muscle was incorporated into the ABM. Each individual cell was represented by an individual agent in the simulation. The network flow model calculates hemodynamic parameters (blood flow rates, fluid shear stress, and hydrostatic pressures) throughout the simulated microvascular network. These are incorporated into the ABM to affect monocyte transit through the network and chemokine/cytokine concentrations. In turn, simulated monocytes respond to their local mechanical and biochemical environments and make behavioral decisions based on a rule set derived from independent literature. Simulated cell behaviors give rise to emergent leukocyte rolling, adhesion, and extravasation. Molecular knockout simulations were performed to validate the model, and predictions of monocyte adhesion, rolling, and extravasation show good agreement with the independently published corresponding mouse studies.

The United States (US) Food and Drug Administration (FDA) is a regulatory agency that has oversight for a wide range of products entering the US market, including gene and cell therapies. The regulatory approach for these products is similar to other medical products within the United States and consists of a multitiered framework of statutes, regulations, and guidance documents. Within this framework, there is considerable flexibility which is necessary due to the biological and technical complexity of these products in general. This chapter provides an overview of the US FDA regulatory oversight of gene and cell therapy products.

The US Food and Drug Administration (FDA) is responsible for the regulatory oversight of a wide range of products, including cell-based products regulated by the Office of Tissues and Advanced Therapies (OTAT) in the Center for Biologics Evaluation and Research. This chapter focuses on several key issues relevant to the development of cell-based products, including early and late phase considerations for chemistry, manufacturing, and controls; pharmacology/toxicology; and clinical testing. This chapter introduces the following topics: the regulatory process, sponsor meetings with OTAT, the requirements for submitting an investigational new drug application (IND), and FDA review of an original IND submission. Also discussed are combination products, tissue-engineered/regenerative medicine products, medical devices, special regulatory considerations such as clinical research involving children, responsibilities of sponsors and investigators, and the use of standards. Knowledge and consideration of the topics discussed in this chapter may aid in the development of cell-based product.

Scientists love a challenge. But for developers of cellular and gene therapy (CGT) products, understanding the scope of preclinical testing needed to support early-phase clinical trials can be daunting. In addition, these diverse and complex products often pose safety concerns that cannot be appropriately evaluated with traditional, standardized approaches to preclinical evaluation of small-molecule drugs and biologics. To help developers address these concerns, the U.S. Food and Drug Administration (FDA) has published a draft guidance for the preclinical assessment of investigational CGT products. This guidance clarifies current regulatory expectations for preclinical information used to support human clinical trials and license application. The draft guidance offers recommendations on the substance and scope of preclinical information needed to support clinical trials for multiple investigational product types, including cellular therapies, gene therapies, therapeutic vaccines, xenotransplantation products, and certain biologic-device combination products (such as cellularized scaffolds) regulated by the Office of Cellular, Tissue, and Gene Therapies (OCTGT). General areas of focus include selection of appropriate animal species and models of disease or injury and the conduct of proof-of-concept and toxicology studies. The guidance also highlights specific safety concerns for CGT products, such as tumorigenic potential for some cellular therapy products, and provides recommendations on approaches to their evaluation. Recommendations are presented in the context of designing preclinical programs that help define the risk-benefit profiles of investigational products in order to justify further testing in humans. This draft guidance, when finalized, will supersede the preclinical recommendations issued in a previous FDA guidance ([www.fda.gov/Drugs/ScienceResearch/ResearchAreas/ucm072987.htm][1]) and is not intended to provide a standardized or “cookbook” approach to the preclinical assessment of investigational CGT products. Rather, this guidance encourages a flexible and science-based approach to preclinical testing in which the kind, duration, and scope of testing are tailored for each product and target clinical indication. In recognizing the importance of product-specific, case-by-case application for the CGT products and the inherent challenges this poses, this guidance encourages early communication between product developers and the OCTGT pharmacology and toxicology review staff. In addition, the guidance emphasizes incorporation of the “3 Rs” principles (reduction, refinement, and replacement of animal use) in preclinical testing programs. Publication of this draft guidance signifies FDA’s continuing effort to engage CGT product developers to help facilitate the safe development of these emerging therapies. Draft Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products; [[Full Text] ][2] [1]: http://www.fda.gov/Drugs/ScienceResearch/ResearchAreas/ucm072987.htm [2]: http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandGeneTherapy/UCM329861.pdf

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published(1-3). For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 μm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

A set of general principles can guide preclinical testing strategies for evaluating the tumorigenicity of regenerative medicine products.

For scientists who have spent much of their time in classrooms and lecture halls, it is second nature to learn by instruction, and Web-based seminars (webinars) or online video tutorials delivered via broadband connections have facilitated learning at home or at the office desk. With the current advances in regenerative medicine, many investigators will for the first time be approaching the U.S. Food and Drug Administration (FDA) with the desire to evaluate their innovative products in clinical trials. Here, we describe new resources to facilitate the path from bench to bedside for translational academic scientists and companies. Indeed, the FDA provides a variety of resources through [www.fda.gov][1] to assist investigators as they navigate the FDA submission process. However, even with these resources, the regulatory process can be challenging, especially for complex products. Regenerative medicine products often consist of a biologic, such as cells, and a device, such as a scaffold made of a synthetic polymer, that may serve as a delivery substrate. For such intricate and resource-intensive products, a detailed understanding of the regulatory process is pivotal to ensure that the transition from discovery to clinical trials is as uneventful as possible. Guidance documents issued by the Center for Devices and Radiological Health (CDRH) and the Center for Biologics Evaluation and Research (CBER) provide scientific and regulatory information. However, regenerative medicine scientists can also benefit from a more tutorial approach. Sensitive to this need, CDRH and CBER’s Office of Cellular, Tissue and Gene Therapies (OCTGT) has developed online video tutorials given by FDA staff who are knowledgeable about regenerative medicine and regulatory science. These tutorials, CDRH Learn and OCTGT Learn , are designed to reach a broad audience in a personalized manner, covering regulatory topics that range from premarket to postmarket aspects of product development under the auspices of these two centers. CDRH Learn presents online video tutorials in English, Spanish, or Chinese and includes topics such as “Overview of Regulatory Requirements: Medical Devices” and “Investigational Device Exemption Process—IDE.” An IDE is a required regulatory submission needed to obtain FDA permission to initiate a clinical trial for a medical device. The OCTGT Learn online video tutorials cover diverse products regulated by OCTGT, including cell and gene therapies and tissue-engineered products. This series focuses on product development to enable initiation of early-phase clinical trials. Topics range from “IND [Investigational New Drug] Basics in OCTGT” to “Preclinical Considerations for Products Regulated in OCTGT.” An IND is a required regulatory submission needed to obtain FDA permission in order to initiate a clinical trial for an investigational pharmaceutical agent. Also offered is a tutorial entitled “Sponsor Meetings with OCTGT,” which provides a step-by-step explanation of various procedures for meeting with this CBER office. This particular tutorial is especially helpful to investigators who are approaching CBER/OCTGT for the first time, because it covers the types of meetings offered, instructions on how to request a meeting, information on what to submit to OCTGT before a meeting, and the type of response investigators can expect from OCTGT during the meeting. This unique online source of comprehensive interactive instruction guides researchers through the regulatory process to encourage the development of promising regenerative medicine therapies. [FDA. Device Advice: Comprehensive Regulatory Assistance. ][2] [FDA. Guidance, Compliance & Regulatory Information (Biologics).][3] [FDA. CDRH Learn. [video tutorial] 12 October 2010.][4] [FDA. OCTGT Learn. [video tutorial] 28 January 2011.][5] [1]: http://www.fda.gov [2]: http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/default.htm [3]: http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/default.htm [4]: http://www.fda.gov/Training/CDRHLearn/default.htm [5]: http://www.fda.gov/BiologicsBloodVaccines/NewsEvents/ucm232821.htm

A growing body of literature suggests that human adipose-derived stromal cells (hASCs) possess developmental plasticity both in vitro and in vivo, and might represent a viable cell source for therapeutic angiogenesis and tissue engineering. We investigate their phenotypic similarity to perivascular cell types, ability to contribute to in vivo microvascular remodeling, and ability to modulate vascular stability. We evaluated hASC surface expression of vascular and stem/progenitor cell markers in vitro, as well as any effects of platelet-derived growth factor B chain (PDGF-BB) and vascular endothelial growth factor 165 on in vitro hASC migration. To ascertain in vivo behavior of hASCs in an angiogenic environment, hASCs were isolated, expanded in culture, labeled with a fluorescent marker, and injected into adult nude rat mesenteries that were stimulated to undergo microvascular remodeling. Ten, 30, and 60 days after injection, tissues from anesthetized animals were harvested and processed with immunohistochemical techniques to determine hASC quantity, positional fate in relation to microvessels, and expression of endothelial and perivascular cell markers. After 60 days, 29% of hASCs exhibited perivascular morphologies compared with 11% of injected human lung fibroblasts. hASCs exhibiting perivascular morphologies also expressed markers characteristic of vascular pericytes: smooth muscle α-actin (10%) and neuron-glia antigen 2 (8%). In tissues treated with hASCs, vascular density was significantly increased over age-matched controls lacking hASCs. This study demonstrates that hASCs express pericyte lineage markers in vivo and in vitro, exhibit increased migration in response to PDGF-BB in vitro, exhibit perivascular morphology when injected in vivo, and contribute to increases in microvascular density during angiogenesis by migrating toward vessels. Disclosure of potential conflicts of interest is found at the end of this article.

A central challenge in the field of developmental biology is to understand how mechanisms at one level of biological scale (i.e., cell-level) interact to produce higher-level (i.e., tissue-level) phenomena. This challenge is particularly relevant to the study of tissue morphogenesis, the process that generates newly formed, remodeled, or regenerated tissue structures. Morphogenesis arises from the spatially- and temporally-dynamic interactions of individual cells with each other and their local environment. Computational models have been combined with experimental efforts to accelerate the discovery processes. Agent-based modeling (ABM) is a computational technique that can be used to model collections of individual biological cells and compute their interactions, which generate emergent tissue-level results. Recently, ABM has been applied to the study of various developmental morphogenic processes, and the purpose of this review is to summarize these studies in order to demonstrate the types of advances that can be expected from pursuing a multicell ABM approach. We also highlight some challenges associated with ABM and suggest strategies for overcoming them. While ABM's application to the study of ecology, epidemiology, and social sciences has a much longer history, we suggest that the application of ABM to the study of morphogenesis has great utility, and when paired with benchtop experimentation, ABM can provide new insights and direct future experimentation. Birth Defects Research (Part C) 81:344–353, 2007. © 2008 Wiley-Liss, Inc.

Intravenous delivery of human adipose-derived stromal cells (hASCs) is a promising option for the treatment of ischemia. After delivery, hASCs that reside and persist in the injured extravascular space have been shown to aid recovery of tissue perfusion and function, although low rates of incorporation currently limit the safety and efficacy of these therapies. We submit that a better understanding of the trafficking of therapeutic hASCs through the microcirculation is needed to address this and that selective control over their homing (organ- and injury-specific) may be possible by targeting bottlenecks in the homing process. This process, however, is incredibly complex, which merited the use of computational techniques to speed the rate of discovery. We developed a multicell agent-based model (ABM) of hASC trafficking during acute skeletal muscle ischemia, based on over 150 literature-based rules instituted in Netlogo and MatLab software programs. In silico, trafficking phenomena within cell populations emerged as a result of the dynamic interactions between adhesion molecule expression, chemokine secretion, integrin affinity states, hemodynamics and microvascular network architectures. As verification, the model reasonably reproduced key aspects of ischemia and trafficking behavior including increases in wall shear stress, upregulation of key cellular adhesion molecules expressed on injured endothelium, increased secretion of inflammatory chemokines and cytokines, quantified levels of monocyte extravasation in selectin knockouts, and circulating monocyte rolling distances. Successful ABM verification prompted us to conduct a series of systematic knockouts in silico aimed at identifying the most critical parameters mediating hASC trafficking. Simulations predicted the necessity of an unknown selectin-binding molecule to achieve hASC extravasation, in addition to any rolling behavior mediated by hASC surface expression of CD15s, CD34, CD62e, CD62p, or CD65. In vitro experiments confirmed this prediction; a subpopulation of hASCs slowly rolled on immobilized P-selectin at speeds as low as 2 µm/s. Thus, our work led to a fundamentally new understanding of hASC biology, which may have important therapeutic implications., Author Summary Ischemic pathologies, such as acute myocardial infarction and peripheral vascular disease, continue to be associated with high morbidities and mortalities. Recently, therapies wherein adult stem cells are injected into the circulation have been shown to increase blood flow and help to restore tissue function following injury. Pre-clinical animal models and human trials have shown successes utilizing this approach, but variable trafficking efficiencies and low incorporation of cells into the injured tissue severely limit effectiveness and may preclude clinical adoption. To address this, we sought to study the complex process of how injected stem cells traffic through the microcirculation and home to sites of injury, in an effort to identify bottlenecks in this process that could be manipulated for therapeutic gain. We developed an agent-based computer model to speed the rate of discovery, and we identified a key cell–cell adhesion interaction that could be targeted to enhance stem cell homing efficiencies during injectable stem cell therapies.