Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes

In many cell types, several cellular processes, such as differentiation of stem/precursor cells, maintenance of differentiated phenotype, motility, adhesion, growth, and survival, strictly depend on the stiffness of extracellular matrix that,in vivo, characterizes their correspondent organ and tissue. In the liver, the stromal rigidity is essential to obtain the correct organ physiology whereas any alteration causes liver cell dysfunctions. The rigidity of the substrate is an element no longer negligible for the cultivation of several cell types, so that many data so far obtained, where cells have been cultured on plastic, could be revised. Regarding liver cells, standard culture conditions lead to the dedifferentiation of primary hepatocytes, transdifferentiation of stellate cells into myofibroblasts, and loss of fenestration of sinusoidal endothelium. Furthermore, standard cultivation of liver stem/precursor cells impedes an efficient execution of the epithelial/hepatocyte differentiation program, leading to the expansion of a cell population expressing only partially liver functions and products. Overcoming these limitations is mandatory for any approach of liver tissue engineering. Here we propose cell lines asin vitromodels of liver stem cells and hepatocytes and an innovative culture method that takes into account the substrate stiffness to obtain, respectively, a rapid and efficient differentiation process and the maintenance of the fully differentiated phenotype.

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Kinases of the Focal Adhesion Complex Contribute to Cardiomyocyte Specification

International Journal of Molecular Sciences ◽

10.3390/ijms221910430 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10430

Author(s):

Sacha Robert ◽

Marcus Flowers ◽

Brenda M. Ogle

Keyword(s):

Stem Cells ◽

Focal Adhesion ◽

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Cell Types ◽

Culture Conditions ◽

Model Systems ◽

Critical Components

Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.

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Adipose Tissue: Understanding the Heterogeneity of Stem Cells for Regenerative Medicine

Biomolecules ◽

10.3390/biom11070918 ◽

2021 ◽

Vol 11 (7) ◽

pp. 918

Author(s):

Wee Kiat Ong ◽

Smarajit Chakraborty ◽

Shigeki Sugii

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Regenerative Medicine ◽

Species Difference ◽

Subcutaneous Fat ◽

Cell Types ◽

Culture Conditions ◽

Cellular Heterogeneity

Adipose-derived stem cells (ASCs) have been increasingly used as a versatile source of mesenchymal stem cells (MSCs) for diverse clinical investigations. However, their applications often become complicated due to heterogeneity arising from various factors. Cellular heterogeneity can occur due to: (i) nomenclature and criteria for definition; (ii) adipose tissue depots (e.g., subcutaneous fat, visceral fat) from which ASCs are isolated; (iii) donor and inter-subject variation (age, body mass index, gender, and disease state); (iv) species difference; and (v) study design (in vivo versus in vitro) and tools used (e.g., antibody isolation and culture conditions). There are also actual differences in resident cell types that exhibit ASC/MSC characteristics. Multilineage-differentiating stress-enduring (Muse) cells and dedifferentiated fat (DFAT) cells have been reported as an alternative or derivative source of ASCs for application in regenerative medicine. In this review, we discuss these factors that contribute to the heterogeneity of human ASCs in detail, and what should be taken into consideration for overcoming challenges associated with such heterogeneity in the clinical use of ASCs. Attempts to understand, define, and standardize cellular heterogeneity are important in supporting therapeutic strategies and regulatory considerations for the use of ASCs.

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A macrophage and theca cell-enriched stromal cell population influences growth and survival of immature murine follicles in vitro

Reproduction ◽

10.1530/rep-10-0483 ◽

2011 ◽

Vol 141 (6) ◽

pp. 809-820 ◽

Cited By ~ 50

Author(s):

Candace M Tingen ◽

Sarah E Kiesewetter ◽

Jennifer Jozefik ◽

Cristina Thomas ◽

David Tagler ◽

...

Keyword(s):

Stromal Cells ◽

Culture System ◽

Stromal Cell ◽

Ovarian Follicle ◽

3D Culture ◽

Culture Conditions ◽

Growth And Survival ◽

3D Culture System

Innovations in in vitro ovarian follicle culture have revolutionized the field of fertility preservation, but the successful culturing of isolated primary and small secondary follicles remains difficult. Herein, we describe a revised 3D culture system that uses a feeder layer of ovarian stromal cells to support early follicle development. This culture system allows significantly improved primary and early secondary follicle growth and survival. The stromal cells, consisting mostly of thecal cells and ovarian macrophages, recapitulate the in vivo conditions of these small follicles and increase the production of androgens and cytokines missing from stromal cell-free culture conditions. These results demonstrate that small follicles have a stage-specific reliance on the ovarian environment, and that growth and survival can be improved in vitro through a milieu created by pre-pubertal ovarian stromal cell co-culture.

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Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515617113 ◽

2015 ◽

Vol 113 (1) ◽

pp. 182-187 ◽

Cited By ~ 124

Author(s):

Christina H. Eng ◽

Zuncai Wang ◽

Diane Tkach ◽

Lourdes Toral-Barza ◽

Savuth Ugwonali ◽

...

Keyword(s):

Anticancer Agents ◽

Kinase Inhibitors ◽

Cell Types ◽

Loss Of Function ◽

Growth And Survival ◽

Oncogenic Mutations ◽

Cellular Context ◽

Genetic Stratification

Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.

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Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening

International Journal of Molecular Sciences ◽

10.3390/ijms21134804 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4804

Author(s):

Vincent van Duinen ◽

Wendy Stam ◽

Eva Mulder ◽

Farbod Famili ◽

Arie Reijerkerk ◽

...

Keyword(s):

Drug Screening ◽

Cell Formation ◽

Cell Types ◽

Culture Conditions ◽

In Vitro Assays ◽

Vegf Receptor ◽

Screening Assay ◽

Angiogenesis Assay

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

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Mesenchymal Stem Cells as a Promising Cell Source for Integration in Novel In Vitro Models

Biomolecules ◽

10.3390/biom10091306 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1306

Author(s):

Ann-Kristin Afflerbach ◽

Mark D. Kiri ◽

Tahir Detinis ◽

Ben M. Maoz

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cell Types ◽

In Vitro Models ◽

Cell Source ◽

Fundamental Research ◽

Multiple Cell ◽

Vitro Model

The human-relevance of an in vitro model is dependent on two main factors—(i) an appropriate human cell source and (ii) a modeling platform that recapitulates human in vivo conditions. Recent years have brought substantial advancements in both these aspects. In particular, mesenchymal stem cells (MSCs) have emerged as a promising cell source, as these cells can differentiate into multiple cell types, yet do not raise the ethical and practical concerns associated with other types of stem cells. In turn, advanced bioengineered in vitro models such as microfluidics, Organs-on-a-Chip, scaffolds, bioprinting and organoids are bringing researchers ever closer to mimicking complex in vivo environments, thereby overcoming some of the limitations of traditional 2D cell cultures. This review covers each of these advancements separately and discusses how the integration of MSCs into novel in vitro platforms may contribute enormously to clinical and fundamental research.

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Human Organoids for Predictive Toxicology Research and Drug Development

Frontiers in Genetics ◽

10.3389/fgene.2021.767621 ◽

2021 ◽

Vol 12 ◽

Author(s):

Toshikatsu Matsui ◽

Tadahiro Shinozawa

Keyword(s):

Stem Cells ◽

Drug Development ◽

Disease Modeling ◽

Cell Types ◽

Underlying Disease ◽

Future Research ◽

Specific Cell ◽

Predictive Toxicology

Organoids are three-dimensional structures fabricated in vitro from pluripotent stem cells or adult tissue stem cells via a process of self-organization that results in the formation of organ-specific cell types. Human organoids are expected to mimic complex microenvironments and many of the in vivo physiological functions of relevant tissues, thus filling the translational gap between animals and humans and increasing our understanding of the mechanisms underlying disease and developmental processes. In the last decade, organoid research has attracted increasing attention in areas such as disease modeling, drug development, regenerative medicine, toxicology research, and personalized medicine. In particular, in the field of toxicology, where there are various traditional models, human organoids are expected to blaze a new path in future research by overcoming the current limitations, such as those related to differences in drug responses among species. Here, we discuss the potential usefulness, limitations, and future prospects of human liver, heart, kidney, gut, and brain organoids from the viewpoints of predictive toxicology research and drug development, providing cutting edge information on their fabrication methods and functional characteristics.

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3D Hepatic Organoid-Based Advancements in LIVER Tissue Engineering

Bioengineering ◽

10.3390/bioengineering8110185 ◽

2021 ◽

Vol 8 (11) ◽

pp. 185

Author(s):

Amit Panwar ◽

Prativa Das ◽

Lay Poh Tan

Keyword(s):

Tissue Engineering ◽

Self Assembly ◽

Liver Tissue ◽

3D Culture ◽

Culture Method ◽

Culture Conditions ◽

Phenotypic Properties ◽

Liver Tissue Engineering

Liver-associated diseases and tissue engineering approaches based on in vitro culture of functional Primary human hepatocytes (PHH) had been restricted by the rapid de-differentiation in 2D culture conditions which restricted their usability. It was proven that cells growing in 3D format can better mimic the in vivo microenvironment, and thus help in maintaining metabolic activity, phenotypic properties, and longevity of the in vitro cultures. Again, the culture method and type of cell population are also recognized as important parameters for functional maintenance of primary hepatocytes. Hepatic organoids formed by self-assembly of hepatic cells are microtissues, and were able to show long-term in vitro maintenance of hepato-specific characteristics. Thus, hepatic organoids were recognized as an effective tool for screening potential cures and modeling liver diseases effectively. The current review summarizes the importance of 3D hepatic organoid culture over other conventional 2D and 3D culture models and its applicability in Liver tissue engineering.

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Vasculogenesis Potential of Mesenchymal and Endothelial Stem Cells Isolated from Various Human Tissues

10.1101/049668 ◽

2016 ◽

Cited By ~ 2

Author(s):

Rokhsareh Rohban ◽

Nathalie Etchart ◽

Thomas R. Pieber

Keyword(s):

Stem Cells ◽

Umbilical Cord ◽

Adult Stem Cell ◽

Cell Types ◽

Human Tissues ◽

Vessel Formation ◽

Nsg Mice ◽

Variable Capacity

AbstractNeo vessel formation can be initiated by co-transplantation of mesenchymal stem cells (MSC) with endothelial colony-forming cells (ECFC). The two adult stem cell types can be isolated and expanded from a variety of tissues to be used for regenerative applications pro-angiogenesis.Here we performed a systematic study to evaluate the neo-vasculogenesis potential of MSC and ECFC isolated from various human tissues. MSC were isolated, purified and expanded in vitro from umbilical cord (UC) and umbilical cord blood (UCB), white adipose tissue (WAT), bone marrow (BM), and amniotic membrane of placenta (AMN).ECFC were isolated from UC and UCB, WAT and peripheral blood (PB). ECFC and MSC and were co-transplanted admixed with extracellular matrix (Matrigel®) at a ratio of 5:1 to immune-deficient NSG mice, subcutaneously. The transplants were harvested after two weeks and the state of vessel formation and stability in the explants were investigated using immune-histochemical methods. The number of created micro-vessels was quantified using Hematoxylin & Eosin (H&E) staining followed by image J quantification.Results showed that ECFC and MSC possess variable capacity in contributing to neo-vasculogenesis. WAT and UCB-derived ECFC and WAT, UCB and BM-derived MSC are most potent cells in terms of neo-vessel formation in vivo. UC-derived ECFC and AMN-derived MSC have been shown to be least potent in contributing to neo-vasculogenesis. This variability might be due to variable phenotypes, or different genetic profiles of MSC and ECFC isolated from different tissues and/or donors.The findings might give an insight into better regenerative strategies for neo-vessel formation in vivo.

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Defining totipotency using criteria of increasing stringency

10.1101/2020.03.02.972893 ◽

2020 ◽

Cited By ~ 7

Author(s):

Eszter Posfai ◽

John Paul Schell ◽

Adrian Janiszewski ◽

Isidora Rovic ◽

Alexander Murray ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Single Cell ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Cell Types ◽

Differentiated Cells ◽

Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

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