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A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Stem Cells".

Deadline for manuscript submissions: closed (30 April 2020) | Viewed by 54471

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Dr. Joni H. Ylostalo

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Department of Biology, University of Mary Hardin-Baylor, 900 College Street, Box 8432, Belton, TX 76513, USA
Interests: mesenchymal stem cell (MSC) biology; 3D-cell culture; microbiology; immunology; regenerative medicine
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Dear Colleagues,

Stem cells have drawn much interest recently because of their unique characteristics and applicability in basic and preclinical research. The use of stem cells in clinical trials has also increased tremendeously, as the cells have shown many promising therapeutic effects in animal models and in patients. Numerous different stem cells have been isolated from various adult and fetal tissues, and these cells are able to self-renew and differentiate into various end-stage cell types. In order to generate enough stem cells for research and therapies, the cells often need to be culture-epxanded. These cultures often employ traditional 2D techniques that do not necessarily mimic the stem cell environment in vivo well. Therefore, great interests lie in developing 3D culture techniques and characterizing stem cells grown under these conditions to understand basic stem cell biology but also develop effective cells for therapies. These 3D cultures might hold the answers for optimal stem cell preparations for clinical applications.

We invite investigators to contribute reviews and original papers describing recent findings in the field of 3D stem cell cultures. We especially encourage submission of research regarding various 3D culture techniques and regenerative medicine approaches using in vitro and in vivo models.

Dr. Joni H. Ylostalo
Guest Editor

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Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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Keywords

stem cell
3D
culture condition
expansion
growth
characteristic
niche
regenerative medicine
animal model

Related Special Issue

3D Stem Cell Culture—Series 2 in Cells (4 articles)

Published Papers (11 papers)

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Editorial

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3 pages, 172 KiB

Open AccessEditorial

3D Stem Cell Culture

by Joni H. Ylostalo

Cells 2020, 9(10), 2178; https://doi.org/10.3390/cells9102178 - 27 Sep 2020

Cited by 15 | Viewed by 4349

Abstract

Much interest has been directed towards stem cells, both in basic and translational research, to understand basic stem cell biology and to develop new therapies for many disorders. In general, stem cells can be cultured with relative ease, however, most common culture methods for stem cells employ 2D techniques using plastic. These cultures do not well represent the stem cell niches in the body, which are delicate microenvironments composed of not only stem cells, but also supporting stromal cells, extracellular matrix, and growth factors. Therefore, researchers and clinicians have been seeking optimal stem cell preparations for basic research and clinical applications, and these might be attainable through 3D culture of stem cells. The 3D cultures recapitulate the in vivo cell-to-cell and cell-to-matrix interactions more effectively, and the cells in 3D cultures exhibit many unique and desirable characteristics. The culture of stem cells in 3D may employ various matrices or scaffolds, in addition to the cells, to support the complex structures. The goal of this Special Issue is to bring together recent research on 3D cultures of various stem cells to increase the basic understanding of stem cells and culture techniques, and also highlight stem cell preparations for possible novel therapeutic applications. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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27 pages, 5749 KiB

Open AccessArticle

Xenobiotic-Free Medium Guarantees Expansion of Adipose Tissue-Derived Canine Mesenchymal Stem Cells Both in 3D Fibrin-Based Matrices and in 2D Plastic Surface Cultures

by Caterina M. Suelzu, Virna Conti, Youssef Khalidy, Sara Montagna, Gabriele Strusi, Rosanna Di Lecce, Priscilla Berni, Giuseppina Basini, Roberto Ramoni and Stefano Grolli

Cells 2020, 9(12), 2578; https://doi.org/10.3390/cells9122578 - 2 Dec 2020

Cited by 8 | Viewed by 2726

Abstract

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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18 pages, 5249 KiB

Open AccessArticle

Activated Cardiac Fibroblasts Control Contraction of Human Fibrotic Cardiac Microtissues by a β-Adrenoreceptor-Dependent Mechanism

by Przemysław Błyszczuk, Christian Zuppinger, Ana Costa, Daria Nurzynska, Franca Di Meglio, Mara Stellato, Irina Agarkova, Godfrey L. Smith, Oliver Distler and Gabriela Kania

Cells 2020, 9(5), 1270; https://doi.org/10.3390/cells9051270 - 20 May 2020

Cited by 9 | Viewed by 4039

Abstract

Cardiac fibrosis represents a serious clinical problem. Development of novel treatment strategies is currently restricted by the lack of the relevant experimental models in a human genetic context. In this study, we fabricated self-aggregating, scaffold-free, 3D cardiac microtissues using human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes and human cardiac fibroblasts. Fibrotic condition was obtained by treatment of cardiac microtissues with profibrotic cytokine transforming growth factor β1 (TGF-β1), preactivation of foetal cardiac fibroblasts with TGF-β1, or by the use of cardiac fibroblasts obtained from heart failure patients. In our model, TGF-β1 effectively induced profibrotic changes in cardiac fibroblasts and in cardiac microtissues. Fibrotic phenotype of cardiac microtissues was inhibited by treatment with TGF-β-receptor type 1 inhibitor SD208 in a dose-dependent manner. We observed that fibrotic cardiac microtissues substantially increased the spontaneous beating rate by shortening the relaxation phase and showed a lower contraction amplitude. Instead, no changes in action potential profile were detected. Furthermore, we demonstrated that contraction of human cardiac microtissues could be modulated by direct electrical stimulation or treatment with the β-adrenergic receptor agonist isoproterenol. However, in the absence of exogenous agonists, the β-adrenoreceptor blocker nadolol decreased beating rate of fibrotic cardiac microtissues by prolonging relaxation time. Thus, our data suggest that in fibrosis, activated cardiac fibroblasts could promote cardiac contraction rate by a direct stimulation of β-adrenoreceptor signalling. In conclusion, a model of fibrotic cardiac microtissues can be used as a high-throughput model for drug testing and to study cellular and molecular mechanisms of cardiac fibrosis. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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21 pages, 4699 KiB

Open AccessArticle

Tendon and Cytokine Marker Expression by Human Bone Marrow Mesenchymal Stem Cells in a Hyaluronate/Poly-Lactic-Co-Glycolic Acid (PLGA)/Fibrin Three-Dimensional (3D) Scaffold

by Maria C. Ciardulli, Luigi Marino, Joseph Lovecchio, Emanuele Giordano, Nicholas R. Forsyth, Carmine Selleri, Nicola Maffulli and Giovanna Della Porta

Cells 2020, 9(5), 1268; https://doi.org/10.3390/cells9051268 - 20 May 2020

Cited by 48 | Viewed by 4182

Abstract

We developed a (three-dimensional) 3D scaffold, we named HY-FIB, incorporating a force-transmission band of braided hyaluronate embedded in a cell localizing fibrin hydrogel and poly-lactic-co-glycolic acid (PLGA) nanocarriers as transient components for growth factor controlled delivery. The tenogenic supporting capacity of HY-FIB on human-Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) was explored under static conditions and under bioreactor-induced cyclic strain conditions. HY-FIB elasticity enabled to deliver a mean shear stress of 0.09 Pa for 4 h/day. Tendon and cytokine marker expression by hBM-MSCs were studied. Results: hBM-MSCs embedded in HY-FIB and subjected to mechanical stimulation, resulted in a typical tenogenic phenotype, as indicated by type 1 Collagen fiber immunofluorescence. RT-qPCR showed an increase of type 1 Collagen, scleraxis, and decorin gene expression (3-fold, 1600-fold, and 3-fold, respectively, at day 11) in dynamic conditions. Cells also showed pro-inflammatory (IL-6, TNF, IL-12A, IL-1β) and anti-inflammatory (IL-10, TGF-β1) cytokine gene expressions, with a significant increase of anti-inflammatory cytokines in dynamic conditions (IL-10 and TGF-β1 300-fold and 4-fold, respectively, at day 11). Mechanical signaling, conveyed by HY-FIB to hBM-MSCs, promoted tenogenic gene markers expression and a pro-repair cytokine balance. The results provide strong evidence in support of the HY-FIB system and its interaction with cells and its potential for use as a predictive in vitro model. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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28 pages, 4471 KiB

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Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres Are Suitable for Neurotoxicity Screening

by Julianna Kobolak, Annamaria Teglasi, Tamas Bellak, Zofia Janstova, Kinga Molnar, Melinda Zana, Istvan Bock, Lajos Laszlo and Andras Dinnyes

Cells 2020, 9(5), 1122; https://doi.org/10.3390/cells9051122 - 1 May 2020

Cited by 32 | Viewed by 7253

Abstract

We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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16 pages, 6580 KiB

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Generation of Differentiating and Long-Living Intestinal Organoids Reflecting the Cellular Diversity of Canine Intestine

by Nina Kramer, Barbara Pratscher, Andre M. C. Meneses, Waltraud Tschulenk, Ingrid Walter, Alexander Swoboda, Hedwig S. Kruitwagen, Kerstin Schneeberger, Louis C. Penning, Bart Spee, Matthias Kieslinger, Sabine Brandt and Iwan A. Burgener

Cells 2020, 9(4), 822; https://doi.org/10.3390/cells9040822 - 28 Mar 2020

Cited by 24 | Viewed by 6068

Abstract

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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15 pages, 7074 KiB

Open AccessArticle

Mouse Embryonic Stem Cell-Derived Ureteric Bud Progenitors Induce Nephrogenesis

by Zenglai Tan, Aleksandra Rak-Raszewska, Ilya Skovorodkin and Seppo J. Vainio

Cells 2020, 9(2), 329; https://doi.org/10.3390/cells9020329 - 31 Jan 2020

Cited by 12 | Viewed by 5287

Abstract

Generation of kidney organoids from pluripotent stem cells (PSCs) is regarded as a potentially powerful way to study kidney development, disease, and regeneration. Direct differentiation of PSCs towards renal lineages is well studied; however, most of the studies relate to generation of nephron progenitor population from PSCs. Until now, differentiation of PSCs into ureteric bud (UB) progenitor cells has had limited success. Here, we describe a simple, efficient, and reproducible protocol to direct differentiation of mouse embryonic stem cells (mESCs) into UB progenitor cells. The mESC-derived UB cells were able to induce nephrogenesis when co-cultured with primary metanephric mesenchyme (pMM). In generated kidney organoids, the embryonic pMM developed nephron structures, and the mESC-derived UB cells formed numerous collecting ducts connected with the nephron tubules. Altogether, our study established an uncomplicated and reproducible platform to generate ureteric bud progenitors from mouse embryonic stem cells. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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19 pages, 4255 KiB

Open AccessArticle

PP2A Deficiency Enhances Carcinogenesis of Lgr5⁺ Intestinal Stem Cells Both in Organoids and In Vivo

by Yu-Ting Yen, May Chien, Yung-Chih Lai, Dao-Peng Chen, Cheng-Ming Chuong, Mien-Chie Hung and Shih-Chieh Hung

Cells 2020, 9(1), 90; https://doi.org/10.3390/cells9010090 - 30 Dec 2019

Cited by 3 | Viewed by 4520

Abstract

In most cancers, cellular origin and the contribution of intrinsic and extrinsic factors toward transformation remain elusive. Cell specific carcinogenesis models are currently unavailable. To investigate cellular origin in carcinogenesis, we developed a tumorigenesis model based on a combination of carcinogenesis and genetically engineered mouse models. We show in organoids that treatment of any of three carcinogens, DMBA, MNU, or PhIP, with protein phosphatase 2A (PP2A) knockout induced tumorigenesis in Lgr5⁺ intestinal lineage, but not in differentiated cells. These transformed cells increased in stem cell signature, were upregulated in EMT markers, and acquired tumorigenecity. A mechanistic approach demonstrated that tumorigenesis was dependent on Wnt, PI3K, and RAS-MAPK activation. In vivo combination with carcinogen and PP2A depletion also led to tumor formation. Using whole-exome sequencing, we demonstrate that these intestinal tumors display mutation landscape and core driver pathways resembling human intestinal tumor in The Cancer Genome Atlas (TCGA). These data provide a basis for understanding the interplay between extrinsic carcinogen and intrinsic genetic modification and suggest that PP2A functions as a tumor suppressor in intestine carcinogenesis. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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17 pages, 5057 KiB

Open AccessArticle

Vertically-Aligned Functionalized Silicon Micropillars for 3D Culture of Human Pluripotent Stem Cell-Derived Cortical Progenitors

by Alessandro Cutarelli, Simone Ghio, Jacopo Zasso, Alessandra Speccher, Giorgina Scarduelli, Michela Roccuzzo, Michele Crivellari, Nicola Maria Pugno, Simona Casarosa, Maurizio Boscardin and Luciano Conti

Cells 2020, 9(1), 88; https://doi.org/10.3390/cells9010088 - 30 Dec 2019

Cited by 20 | Viewed by 3910

Abstract

Silicon is a promising material for tissue engineering since it allows to produce micropatterned scaffolding structures resembling biological tissues. Using specific fabrication methods, it is possible to build aligned 3D network-like structures. In the present study, we exploited vertically-aligned silicon micropillar arrays as culture systems for human iPSC-derived cortical progenitors. In particular, our aim was to mimic the radially-oriented cortical radial glia fibres that during embryonic development play key roles in controlling the expansion, radial migration and differentiation of cortical progenitors, which are, in turn, pivotal to the establishment of the correct multilayered cerebral cortex structure. Here we show that silicon vertical micropillar arrays efficiently promote expansion and stemness preservation of human cortical progenitors when compared to standard monolayer growth conditions. Furthermore, the vertically-oriented micropillars allow the radial migration distinctive of cortical progenitors in vivo. These results indicate that vertical silicon micropillar arrays can offer an optimal system for human cortical progenitors’ growth and migration. Furthermore, similar structures present an attractive platform for cortical tissue engineering. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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20 pages, 5997 KiB

Open AccessArticle

Self-Assembling Scaffolds Supported Long-Term Growth of Human Primed Embryonic Stem Cells and Upregulated Core and Naïve Pluripotent Markers

by Christina McKee, Christina Brown and G. Rasul Chaudhry

Cells 2019, 8(12), 1650; https://doi.org/10.3390/cells8121650 - 16 Dec 2019

Cited by 8 | Viewed by 5106

Abstract

The maintenance and expansion of human embryonic stem cells (ESCs) in two-dimensional (2-D) culture is technically challenging, requiring routine manipulation and passaging. We developed three-dimensional (3-D) scaffolds to mimic the in vivo microenvironment for stem cell proliferation. The scaffolds were made of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups, which self-assembled via a Michael addition reaction. When primed ESCs (H9 cells) were mixed with PEG polymers, they were encapsulated and grew for an extended period, while maintaining their viability, self-renewal, and differentiation potential both in vitro and in vivo. Three-dimensional (3-D) self-assembling scaffold-grown cells displayed an upregulation of core pluripotency genes, OCT4, NANOG, and SOX2. In addition, the expression of primed markers decreased, while the expression of naïve markers substantially increased. Interestingly, the expression of mechanosensitive genes, YAP and TAZ, was also upregulated. YAP inhibition by Verteporfin abrogated the increased expression of YAP/TAZ as well as core and naïve pluripotent markers. Evidently, the 3-D culture conditions induced the upregulation of makers associated with a naïve state of pluripotency in the primed cells. Overall, our 3-D culture system supported the expansion of a homogenous population of ESCs and should be helpful in advancing their use for cell therapy and regenerative medicine. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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Review

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13 pages, 863 KiB

Open AccessReview

Traditional and Advanced Cell Cultures in Hematopoietic Stem Cell Studies

by Antonio Carlos Ribeiro-Filho, Débora Levy, Jorge Luis Maria Ruiz, Marluce da Cunha Mantovani and Sérgio Paulo Bydlowski

Cells 2019, 8(12), 1628; https://doi.org/10.3390/cells8121628 - 12 Dec 2019

Cited by 16 | Viewed by 5750

Abstract

Hematopoiesis is the main function of bone marrow. Human hematopoietic stem and progenitor cells reside in the bone marrow microenvironment, making it a hotspot for the development of hematopoietic diseases. Numerous alterations that correspond to disease progression have been identified in the bone marrow stem cell niche. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells determine the balance between the proliferation, differentiation and homeostasis of the stem cell compartment. Changes in this tightly regulated network can provoke malignant transformation. However, our understanding of human hematopoiesis and the associated niche biology remains limited due to accessibility to human material and the limits of in vitro culture models. Traditional culture systems for human hematopoietic studies lack microenvironment niches, spatial marrow gradients, and dense cellularity, rendering them incapable of effectively translating marrow physiology ex vivo. This review will discuss the importance of 2D and 3D culture as a physiologically relevant system for understanding normal and abnormal hematopoiesis. Full article

(This article belongs to the Special Issue 3D Stem Cell Culture)

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