Problems and prospects for the use of stem cells in transplantation

The problems of organ and tissue transplantation are the lack of organs for transplantation and the rejection of transplants. Therefore, the issue of obtaining organs and tissues for transplantation with stem cells is being studied. Although this idea is promising, it is associated with many problems. To do this, you need to use several populations of cells on a substrate with a complex composition of nutrient environments: nutrients, growth factors, oxygen, regulatory factors. Intercellular interaction is provided by the factors they secrete, or it occurs directly with intercellular contact. This contributes to the fact that stem cells in test tubes can differentiate into other types of tissues and maintain their biological activity indefinitely, which they cannot in vivo. This approach of tissue engineering provides the possibility of obtaining whole organs for implantation. However, technical problems are associated with increased cell adhesion to plastic, the presence of a universal basis for cell nutrition, which can contain more than 100 components. There is a possibility of contamination, which can lead to serious errors in the experiment. Stem cells must have distinct mutational properties and the ability to restore telome cells. Prolonged use of the same nutrient medium can lead to genetic changes and significantly alter the physiological properties of cells. Cryopreservation can be an important aspect of the solution. The goal of tissue bioengineering is to create whole artificial organs, or at least areas of organized tissue that could be transplanted to patients. Currently, such operations are relatively simple for tissues such as artificial skin consisting of epidermal and fibroblast layers, or small cartilage implants obtained in vitro. Several cell types in stable shape are planned to be used in one environment. In this case, one type of cell can be replaced by another. This stability is provided by a variety of secreted factors by different types of cells that ensure their vitality. Decellularization removes all components involved in immune rejection of grafts, so this raises the prospect of creating an unlimited supply of organs for transplantation. However, acute reactions can develop associated with the participation of dendritic cells, macrophages, neutrophils, natural killers. Starting from the moment of transplantation, conditions for immune rejection are created, arising as a result of surgery with the development of acute inflammation. The intensity of immune reactions against the graft largely depends on the degree of non-conformity of alleles of the main complex of histocompany capacity of the donor and recipient. This match is studied using a variety of methods, including the use of antibodies or sequencing of deoxyribonucleic acid.

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Chimeric RNA:DNA Donorguide Improves HDR in vitro and in vivo

10.1101/2021.05.28.446234 ◽

2021 ◽

Author(s):

Brandon W Simone ◽

Han B Lee ◽

Camden L Daby ◽

Santiago Restrepo-Castillo ◽

Hirotaka Ata ◽

...

Keyword(s):

Cell Types ◽

Repair Process ◽

Strand Break ◽

Genetic Alterations ◽

Precise Integration ◽

Genetic Changes ◽

Area Of Interest ◽

Temporal Localization

Introducing small genetic changes to study specific mutations or reverting clinical mutations to wild type has been an area of interest in precision genomics for several years. In fact, it has been found that nearly 90% of all human pathogenic mutations are caused by small genetic variations, and the methods to efficiently and precisely correct these errors are critically important. One common way to make these small DNA changes is to provide a single stranded DNA (ssDNA) donor containing the desired alteration together with a targeted double-strand break (DSB) at the genomic target. The donor is typically flanked by regions of homology that are often ~30-100bp in length to leverage the homology directed repair (HDR) pathway. Coupling a ssDNA donor with a CRISPR-Cas9 to produce a targeted DSB is one of the most streamlined approaches to introduce small changes. However, in many cell types this approach results in a low rate of incorporation of the desired alteration and has undesired imprecise repair at the 5' or 3' junction due to artifacts of the DNA repair process. We herein report a technology that couples the spatial temporal localization of an ssDNA repair template and leverages the nucleic acid components of the CRISPR-Cas9 system. We show that by direct fusion of an ssDNA template to the trans activating RNA (tracrRNA) to generate an RNA-DNA chimera, termed Donorguide, we recover precise integration of genetic alterations, with both increased integration rates and decreased imprecision at the 5' or 3' junctions relative to an ssODN donor in vitro in HEK293T cells as well as in vivo in zebrafish. Further, we show that this technology can be used to enhance gene conversion with other gene editing tools such as TALENs.

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

Stem Cells International ◽

10.1155/2016/5481493 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 38

Author(s):

Angela Maria Cozzolino ◽

Valeria Noce ◽

Cecilia Battistelli ◽

Alessandra Marchetti ◽

Germana Grassi ◽

...

Keyword(s):

Stem Cells ◽

Culture Method ◽

Cell Types ◽

Culture Conditions ◽

Precursor Cells ◽

Substrate Stiffness ◽

Growth And Survival ◽

Liver Stem Cells

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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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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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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Constricted migration modulates stem cell differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e19-02-0090 ◽

2019 ◽

Vol 30 (16) ◽

pp. 1985-1999 ◽

Cited By ~ 7

Author(s):

Lucas R. Smith ◽

Jerome Irianto ◽

Yuntao Xia ◽

Charlotte R. Pfeifer ◽

Dennis E. Discher

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Stem Cell Differentiation ◽

Cell Types ◽

Human Mscs ◽

Nuclear Forces ◽

Nuclear Entry ◽

Nuclear Rupture

Tissue regeneration at an injured site depends on proliferation, migration, and differentiation of resident stem or progenitor cells, but solid tissues are often sufficiently dense and constricting that nuclei are highly stressed by migration. In this study, constricted migration of myoblastic cell types and mesenchymal stem cells (MSCs) increases nuclear rupture, increases DNA damage, and modulates differentiation. Fewer myoblasts fuse into regenerating muscle in vivo after constricted migration in vitro, and myodifferentiation in vitro is likewise suppressed. Myosin II inhibition rescues rupture and DNA damage, implicating nuclear forces, while mitosis and the cell cycle are suppressed by constricted migration, consistent with a checkpoint. Although perturbed proliferation fails to explain defective differentiation, nuclear rupture mislocalizes differentiation-relevant MyoD and KU80 (a DNA repair factor), with nuclear entry of the DNA-binding factor cGAS. Human MSCs exhibit similar damage, but osteogenesis increases—which is relevant to bone and to calcified fibrotic tissues, including diseased muscle. Tissue repair can thus be modulated up or down by the curvature of pores through which stem cells squeeze.

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Reversine: A Synthetic Purine with a Dual Activity as a Cell Dedifferentiating Agent and a Selective Anticancer Drug

Current Medicinal Chemistry ◽

10.2174/0929867326666190103120725 ◽

2020 ◽

Vol 27 (21) ◽

pp. 3448-3462

Author(s):

Marco Piccoli ◽

Andrea Ghiroldi ◽

Michelle M. Monasky ◽

Federica Cirillo ◽

Giuseppe Ciconte ◽

...

Keyword(s):

Stem Cells ◽

Current Knowledge ◽

Embryonic Stem ◽

Cell Types ◽

Cancer Drug ◽

Anti Cancer ◽

Adult Cell ◽

Induced Pluripotent

The development of new therapeutic applications for adult and embryonic stem cells has dominated regenerative medicine and tissue engineering for several decades. However, since 2006, induced Pluripotent Stem Cells (iPSCs) have taken center stage in the field, as they promised to overcome several limitations of the other stem cell types. Nonetheless, other promising approaches for adult cell reprogramming have been attempted over the years, even before the generation of iPSCs. In particular, two years before the discovery of iPSCs, the possibility of synthesizing libraries of large organic compounds, as well as the development of high-throughput screenings to quickly test their biological activity, enabled the identification of a 2,6-disubstituted purine, named reversine, which was shown to be able to reprogram adult cells to a progenitor-like state. Since its discovery, the effect of reversine has been confirmed on different cell types, and several studies on its mechanism of action have revealed its central role in inhibitory activity on several kinases implicated in cell cycle regulation and cytokinesis. These key features, together with its chemical nature, suggested a possible use of the molecule as an anti-cancer drug. Remarkably, reversine exhibited potent cytotoxic activity against several tumor cell lines in vitro and a significant effect in decreasing tumor progression and metastatization in vivo. Thus, 15 years since its discovery, this review aims at critically summarizing the current knowledge to clarify the dual role of reversine as a dedifferentiating agent and anti-cancer drug.

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Partial Reprogramming As An Emerging Strategy for Safe Induced Cell Generation and Rejuvenation

Current Gene Therapy ◽

10.2174/1566523219666190902154511 ◽

2019 ◽

Vol 19 (4) ◽

pp. 248-254

Author(s):

Marianne Lehmann ◽

Martina Canatelli-Mallat ◽

Priscila Chiavellini ◽

Gloria M. Cónsole ◽

Maria D. Gallardo ◽

...

Keyword(s):

Stem Cells ◽

Somatic Cell ◽

Fluorescent Protein ◽

Somatic Cells ◽

Cell Types ◽

Cell Reprogramming ◽

Green Fluorescent ◽

Original Cell

Background: Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research. Objective: Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation. Methods: In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM). Results: Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter. Conclusion: Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.

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Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning

eLife ◽

10.7554/elife.32839 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 73

Author(s):

Sophie M Morgani ◽

Jakob J Metzger ◽

Jennifer Nichols ◽

Eric D Siggia ◽

Anna-Katerina Hadjantonakis

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Pluripotent Stem Cells ◽

Epithelial To Mesenchymal Transition ◽

Cell Types ◽

Mesenchymal Transition ◽

Fate Specification ◽

Epiblast Stem Cells

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species.

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