Strategies for genetic manipulation of adult stem cell-derived organoids

AbstractOrganoid technology allows the expansion of primary epithelial cells from normal and diseased tissues, providing a unique model for human (patho)biology. In a three-dimensional environment, adult stem cells self-organize and differentiate to gain tissue-specific features. Accessibility to genetic manipulation enables the investigation of the molecular mechanisms underlying cell fate regulation, cell differentiation and cell interactions. In recent years, powerful methodologies using lentiviral transgenesis, CRISPR/Cas9 gene editing, and single-cell readouts have been developed to study gene function and carry out genetic screens in organoids. However, the multicellularity and dynamic nature of stem cell-derived organoids also present challenges for genetic experimentation. In this review, we focus on adult gastrointestinal organoids and summarize the state-of-the-art protocols for successful transgenesis. We provide an outlook on emerging genetic techniques that could further increase the applicability of organoids and enhance the potential of organoid-based techniques to deepen our understanding of gene function in tissue biology.

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Stem cell mediated cardiovascular repair

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y2012-010 ◽

2012 ◽

Vol 90 (3) ◽

pp. 337-351 ◽

Cited By ~ 2

Author(s):

Serkan Durdu ◽

Gunseli Cubukcuoglu Deniz ◽

Arin Dogan ◽

Cagin Zaim ◽

Aynur Karadag ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Progenitor Cell ◽

Adult Stem Cells ◽

Molecular Mechanisms ◽

Therapeutic Potential ◽

Imaging Techniques ◽

Developed Countries ◽

Health Issues

Recent increase in the interest in stem and progenitor cells may be attributed to their behavioural characteristics. A consensus has been reached that embryonic or adult stem cells have therapeutic potential. As cardiovascular health issues are still the major culprits in many developed countries, stem and progenitor cell driven approaches may give the clinicians a new arsenal to tackle many significant health issues. However, stem and progenitor cell mediated cardiovascular regeneration can be achieved via complex and dynamic molecular mechanisms involving a variety of cells, growth factors, cytokines, and genes. Functional contributions of transplanted cells on target organs and their survival are still critical problems waiting to be resolved. Moreover, the regeneration of contracting myocardial tissue has controversial results in human trials. Thus, moderately favourable clinical results should be interpreted carefully. Determining the behavioural programs, genetic and transcriptional control of stem cells, mechanisms that determine cell fate, and functional characteristics are the primary targets. In addition, ensuring the long-term follow-up of cells with efficient imaging techniques in human clinical studies may provide a resurgence of the initial enthusiasm, which has faded over time. Here, we provide a brief historical perspective on stem cell driven cardiac regeneration and discuss cardiac and vascular repair in the context of translational science.

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Tissue Regeneration without Stem Cell Transplantation: Self-Healing Potential from Ancestral Chemistry and Physical Energies

Stem Cells International ◽

10.1155/2018/7412035 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Federica Facchin ◽

Eva Bianconi ◽

Silvia Canaider ◽

Valentina Basoli ◽

Pier Mario Biava ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Electromagnetic Fields ◽

Tissue Regeneration ◽

Adult Stem Cells ◽

Molecular Mechanisms ◽

The Body ◽

Self Healing ◽

Mechanical Vibrations

The human body constantly regenerates after damage due to the self-renewing and differentiating properties of its resident stem cells. To recover the damaged tissues and regenerate functional organs, scientific research in the field of regenerative medicine is firmly trying to understand the molecular mechanisms through which the regenerative potential of stem cells may be unfolded into a clinical application. The finding that some organisms are capable of regenerative processes and the study of conserved evolutionary patterns in tissue regeneration may lead to the identification of natural molecules of ancestral species capable to extend their regenerative potential to human tissues. Such a possibility has also been strongly suggested as a result of the use of physical energies, such as electromagnetic fields and mechanical vibrations in human adult stem cells. Results from scientific studies on stem cell modulation confirm the possibility to afford a chemical manipulation of stem cell fate in vitro and pave the way to the use of natural molecules, as well as electromagnetic fields and mechanical vibrations to target human stem cells in their niche inside the body, enhancing human natural ability for self-healing.

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GSK3β, a Master Kinase in the Regulation of Adult Stem Cell Behavior

Cells ◽

10.3390/cells10020225 ◽

2021 ◽

Vol 10 (2) ◽

pp. 225

Author(s):

Claire Racaud-Sultan ◽

Nathalie Vergnolle

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Adult Stem Cells ◽

Glycogen Synthase ◽

Inflammatory Diseases ◽

Adult Stem Cell ◽

Leukemic Cells ◽

Cell Phenotype ◽

Epithelial Regeneration ◽

Cell Functions

In adult stem cells, Glycogen Synthase Kinase 3β (GSK3β) is at the crossroad of signaling pathways controlling survival, proliferation, adhesion and differentiation. The microenvironment plays a key role in the regulation of these cell functions and we have demonstrated that the GSK3β activity is strongly dependent on the engagement of integrins and protease-activated receptors (PARs). Downstream of the integrin α5β1 or PAR2 activation, a molecular complex is organized around the scaffolding proteins RACK1 and β-arrestin-2 respectively, containing the phosphatase PP2A responsible for GSK3β activation. As a consequence, a quiescent stem cell phenotype is established with high capacities to face apoptotic and metabolic stresses. A protective role of GSK3β has been found for hematopoietic and intestinal stem cells. Latters survived to de-adhesion through PAR2 activation, whereas formers were protected from cytotoxicity through α5β1 engagement. However, a prolonged activation of GSK3β promoted a defect in epithelial regeneration and a resistance to chemotherapy of leukemic cells, paving the way to chronic inflammatory diseases and to cancer resurgence, respectively. In both cases, a sexual dimorphism was measured in GSK3β-dependent cellular functions. GSK3β activity is a key marker for inflammatory and cancer diseases allowing adjusted therapy to sex, age and metabolic status of patients.

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Modernized Tools for Streamlined Genetic Manipulation and Comparative Study of Wild and Diverse Proteobacterial Lineages

mBio ◽

10.1128/mbio.01877-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 24

Author(s):

Travis J. Wiles ◽

Elena S. Wall ◽

Brandon H. Schlomann ◽

Edouard A. Hay ◽

Raghuveer Parthasarathy ◽

...

Keyword(s):

Comparative Study ◽

Molecular Mechanisms ◽

Genetic Manipulation ◽

Functional Characterization ◽

Symbiotic Bacteria ◽

Bacterial Isolates ◽

Major Barrier ◽

Allelic Exchange ◽

Genetic Techniques

ABSTRACTCorrelating the presence of bacteria and the genes they carry with aspects of plant and animal biology is rapidly outpacing the functional characterization of naturally occurring symbioses. A major barrier to mechanistic studies is the lack of tools for the efficient genetic manipulation of wild and diverse bacterial isolates. To address the need for improved molecular tools, we used a collection of proteobacterial isolates native to the zebrafish intestinal microbiota as a testbed to construct a series of modernized vectors that expedite genetic knock-in and knockout procedures across lineages. The innovations that we introduce enhance the flexibility of conventional genetic techniques, making it easier to manipulate many different bacterial isolates with a single set of tools. We developed alternative strategies for domestication-free conjugation, designed plasmids with customizable features, and streamlined allelic exchange using visual markers of homologous recombination. We demonstrate the potential of these tools through a comparative study of bacterial behavior within the zebrafish intestine. Live imaging of fluorescently tagged isolates revealed a spectrum of distinct population structures that differ in their biogeography and dominant growth mode (i.e., planktonic versus aggregated). Most striking, we observed divergent genotype-phenotype relationships: several isolates that are predicted by genomic analysis andin vitroassays to be capable of flagellar motility do not display this trait within living hosts. Together, the tools generated in this work provide a new resource for the functional characterization of wild and diverse bacterial lineages that will help speed the research pipeline from sequencing-based correlations to mechanistic underpinnings.IMPORTANCEA great challenge in microbiota research is the immense diversity of symbiotic bacteria with the capacity to impact the lives of plants and animals. Moving beyond correlative DNA sequencing-based studies to define the cellular and molecular mechanisms by which symbiotic bacteria influence the biology of their hosts is stalling because genetic manipulation of new and uncharacterized bacterial isolates remains slow and difficult with current genetic tools. Moreover, developing tools de novo is an arduous and time-consuming task and thus represents a significant barrier to progress. To address this problem, we developed a suite of engineering vectors that streamline conventional genetic techniques by improving postconjugation counterselection, modularity, and allelic exchange. Our modernized tools and step-by-step protocols will empower researchers to investigate the inner workings of both established and newly emerging models of bacterial symbiosis.

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Article Commentary: Stem Cell Research in Cell Transplantation: An Analysis of Geopolitical Influence by Publications

Cell Transplantation ◽

10.3727/000000007783465190 ◽

2007 ◽

Vol 16 (8) ◽

pp. 867-873 ◽

Cited By ~ 5

Author(s):

David J. Eve ◽

Paul R. Sanberg

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Transplantation ◽

Adult Stem Cells ◽

Adult Stem Cell ◽

Therapeutic Area ◽

Fetal Stem Cells ◽

Major Player ◽

The Us ◽

Restricted Use

One of the fastest growing fields in researching treatments for neurodegenerative and other disorders is the use of stem cells. These cells are naturally occurring and can be obtained from three different stages of an organism's life: embryonic, fetal, and adult. In the US, political doctrine has restricted use of federal funds for stem cells, enhancing research towards an adult source. In order to determine how this legislation may be represented by the stem cell field, a retrospective analysis of stem cell articles published in the journal Cell Transplantation over a 2-year period was performed. Cell Transplantation is considered a translational journal from preclinical to clinical, so it was of interest to determine the publication outcome of stem cell articles 6 years after the US regulations. The distribution of the source of stem cells was found to be biased towards the adult stage, but relatively similar over the embryonic and fetal stages. The fetal stem cell reports were primarily neural in origin, whereas the adult stem cell ones were predominantly mesenchymal and used mainly in neural studies. The majority of stem cell studies published in Cell Transplantation were found to fall under the umbrella of neuroscience research. American scientists published the most articles using stem cells with a bias towards adult stem cells, supporting the effect of the legislation, whereas Europe was the leading continent with a bias towards embryonic and fetal stem cells, where research is “controlled” but not restricted. Japan was also a major player in the use of stem cells. Allogeneic transplants (where donor and recipient are the same species) were the most common transplants recorded, although the transplantation of human-derived stem cells into rodents was the most common specific transplantation performed. This demonstrates that the use of stem cells is an increasingly important field (with a doubling of papers between 2005 and 2006), which is likely to develop into a major therapeutic area over the next few decades and that funding restrictions can affect the type of research being performed.

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The Winding Road of Cardiac Regeneration—Stem Cell Omics in the Spotlight

Cells ◽

10.3390/cells7120255 ◽

2018 ◽

Vol 7 (12) ◽

pp. 255 ◽

Cited By ~ 4

Author(s):

Miruna Mihaela Micheu ◽

Alina Ioana Scarlatescu ◽

Alexandru Scafa-Udriste ◽

Maria Dorobantu

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Cell Biology ◽

Molecular Mechanisms ◽

Unmet Need ◽

Cardiac Regeneration ◽

Cell Types ◽

Great Promise ◽

Omics Data

Despite significant progress in treating ischemic cardiac disease and succeeding heart failure, there is still an unmet need to develop effective therapeutic strategies given the persistent high-mortality rate. Advances in stem cell biology hold great promise for regenerative medicine, particularly for cardiac regeneration. Various cell types have been used both in preclinical and clinical studies to repair the injured heart, either directly or indirectly. Transplanted cells may act in an autocrine and/or paracrine manner to improve the myocyte survival and migration of remote and/or resident stem cells to the site of injury. Still, the molecular mechanisms regulating cardiac protection and repair are poorly understood. Stem cell fate is directed by multifaceted interactions between genetic, epigenetic, transcriptional, and post-transcriptional mechanisms. Decoding stem cells’ “panomic” data would provide a comprehensive picture of the underlying mechanisms, resulting in patient-tailored therapy. This review offers a critical analysis of omics data in relation to stem cell survival and differentiation. Additionally, the emerging role of stem cell-derived exosomes as “cell-free” therapy is debated. Last but not least, we discuss the challenges to retrieve and analyze the huge amount of publicly available omics data.

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HMGA Proteins in Stemness and Differentiation of Embryonic and Adult Stem Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21010362 ◽

2020 ◽

Vol 21 (1) ◽

pp. 362 ◽

Cited By ~ 9

Author(s):

Silvia Parisi ◽

Silvia Piscitelli ◽

Fabiana Passaro ◽

Tommaso Russo

Keyword(s):

Experimental Data ◽

Stem Cells ◽

Cell Fate ◽

Adult Stem Cells ◽

Regulation Of Gene Expression ◽

Embryonic Stem ◽

Adult Stem Cell ◽

Mouse Development ◽

Architectural Proteins ◽

Numerous Experimental Data

HMGA1 and HMGA2 are chromatin architectural proteins that do not have transcriptional activity per se, but are able to modify chromatin structure by interacting with the transcriptional machinery and thus negatively or positively regulate the transcription of several genes. They have been extensively studied in cancer where they are often found to be overexpressed but their functions under physiologic conditions have still not been completely addressed. Hmga1 and Hmga2 are expressed during the early stages of mouse development, whereas they are not detectable in most adult tissues. Hmga overexpression or knockout studies in mouse have pointed to a key function in the development of the embryo and of various tissues. HMGA proteins are expressed in embryonic stem cells and in some adult stem cells and numerous experimental data have indicated that they play a fundamental role in the maintenance of stemness and in the regulation of differentiation. In this review, we discuss available experimental data on HMGA1 and HMGA2 functions in governing embryonic and adult stem cell fate. Moreover, based on the available evidence, we will aim to outline how HMGA expression is regulated in different contexts and how these two proteins contribute to the regulation of gene expression and chromatin architecture in stem cells.

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Dynamic Mechanical Properties Control Adult Stem Cell Fate

ASME 2012 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2012-80062 ◽

2012 ◽

Author(s):

Murat Guvendiren ◽

Jason A. Burdick

Keyword(s):

Stem Cell ◽

Cell Fate ◽

Adult Stem Cell ◽

Dynamic Mechanical Properties ◽

Cellular Functions ◽

Cellular Processes ◽

Proliferation And Differentiation ◽

Important Field ◽

Control Adult

Stem cells respond to many microenvironmental cues towards their decisions to spread, migrate, and differentiate and these cues can be incorporated into materials for regenerative medicine.1 In the last decade, matrix stiffness alone has been implicated in regulating cellular functions such as migration, proliferation and differentiation. With this in mind, a variety of natural and synthetic polymer systems were used in vitro to mimic the elasticity of native tissues. Despite helping to develop this important field and gather valuable information, these substrates are primarily static and lack the dynamic nature that is observed during many cellular processes such as development, fibrosis and cancer. Thus, it is of great interest to temporally manipulate matrix elasticity in vitro to better understand and develop strategies to control these biological processes. In this work, we utilize a sequential crosslinking approach (initial gelation via addition reaction, secondary crosslinking through light-mediated radical polymerization) to fabricate hydrogel substrates that stiffen (e.g., ∼3 to 30 kPa) either immediately or at later times and in the presence of cells. We demonstrate the utility of this technique by investigating the short-term (several minutes to hours) and long-term (several days to weeks) stem cell response to dynamic stiffening

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Alternative polyadenylation of Pax3 controls muscle stem cell fate and muscle function

Science ◽

10.1126/science.aax1694 ◽

2019 ◽

Vol 366 (6466) ◽

pp. 734-738 ◽

Cited By ~ 11

Author(s):

Antoine de Morree ◽

Julian D. D. Klein ◽

Qiang Gan ◽

Jean Farup ◽

Andoni Urtasun ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Messenger Rna ◽

Adult Stem Cells ◽

Alternative Polyadenylation ◽

Quiescent State ◽

Stem Cell Fate ◽

Activation Rate

Adult stem cells are essential for tissue homeostasis. In skeletal muscle, muscle stem cells (MuSCs) reside in a quiescent state, but little is known about the mechanisms that control homeostatic turnover. Here we show that, in mice, the variation in MuSC activation rate among different muscles (for example, limb versus diaphragm muscles) is determined by the levels of the transcription factor Pax3. We further show that Pax3 levels are controlled by alternative polyadenylation of its transcript, which is regulated by the small nucleolar RNA U1. Isoforms of the Pax3 messenger RNA that differ in their 3′ untranslated regions are differentially susceptible to regulation by microRNA miR206, which results in varying levels of the Pax3 protein in vivo. These findings highlight a previously unrecognized mechanism of the homeostatic regulation of stem cell fate by multiple RNA species.

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SOX2 for Stem Cell Therapy and Medical Use: Pros or Cons?

Cell Transplantation ◽

10.1177/0963689720907565 ◽

2020 ◽

Vol 29 ◽

pp. 096368972090756

Author(s):

Hong-Meng Chuang ◽

Mao-Hsuan Huang ◽

Yu-Shuan Chen ◽

Horng-Jyh Harn

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Fate ◽

Cell Transplantation ◽

Molecular Mechanisms ◽

Control Cell ◽

Critical Pathways ◽

Stem Cell Isolation ◽

And Storage

Stem cell transplantation is a fast-developing technique, which includes stem cell isolation, purification, and storage, and it is in high demand in the industry. In addition, advanced applications of stem cell transplantation, including differentiation, gene delivery, and reprogramming, are presently being studied in clinical trials. In contrast to somatic cells, stem cells are self-renewing and have the ability to differentiate; however, the molecular mechanisms remain unclear. SOX2 (sex-determining region Y [ SRY]-b ox 2) is one of the well-known reprogramming factors, and it has been recognized as an oncogene associated with cancer induction. The exclusion of SOX2 in reprogramming methodologies has been used as an alternative cancer treatment approach. However, the manner by which SOX2 induces oncogenic effects remains unclear, with most studies demonstrating its regulation of the cell cycle and no insight into the maintenance of cellular stemness. For controlling certain critical pathways, including Shh and Wnt pathways, SOX2 is considered irreplaceable and is required for the normal functioning of stem cells, particularly neural stem cells. In this report, we discussed the functions of SOX2 in both stem and cancer cells, as well as how this powerful regulator can be used to control cell fate.

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