Directed differentiation

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Directed differentiation is a bioengineering methodology at the interface of stem cell biology, developmental biology and tissue engineering. [1] It is essentially harnessing the potential of stem cells by constraining their differentiation in vitro toward a specific cell type or tissue of interest. [2] Stem cells are by definition pluripotent, able to differentiate into several cell types such as neurons, [3] cardiomyocytes, hepatocytes, etc. Efficient directed differentiation requires a detailed understanding of the lineage and cell fate decision, often provided by developmental biology. [2] [4]

Contents

Conceptual frame

During differentiation, pluripotent cells make a number of developmental decisions to generate first the three germ layers (ectoderm, mesoderm and endoderm) of the embryo and intermediate progenitors, [5] followed by subsequent decisions or check points, giving rise to all the body's mature tissues. [4] The differentiation process can be modeled as sequence of binary decisions based on probabilistic or stochastic models. Developmental biology and embryology provides the basic knowledge of the cell types' differentiation through mutation analysis, lineage tracing, embryo micro-manipulation and gene expression studies. Cell differentiation and tissue organogenesis involve a limited set of developmental signaling pathways. [4] It is thus possible to direct cell fate by controlling cell decisions through extracellular signaling, mimicking developmental signals.

Source material

Directed differentiation is primarily applied to pluripotent stem cells (PSCs) of mammalian origin, in particular mouse and human cells for biomedical research applications. [5] Since the discovery of embryonic stem (ES) cells (1981) and induced pluripotent stem (iPS) cells (2006), source material is potentially unlimited. [1] [4] [6] Historically, embryonic carcinoma (EC) cells have also been used. [7] Fibroblasts or other differentiated cell types have been used for direct reprogramming strategies. [1]

Methods

Cell differentiation involves a transition from a proliferative mode toward differentiation mode. Directed differentiation consists in mimicking developmental (embryo's development) decisions in vitro using the stem cells as source material. [1] For this purpose, pluripotent stem cells (PSCs) are cultured in controlled conditions involving specific substrate or extracellular matrices promoting cell adhesion and differentiation, and define culture media compositions. [4] A limited number of signaling factors such as growth factors or small molecules, controlling cell differentiation, is applied sequentially or in a combinatorial manner, at varying dosage and exposure time. [1] Proper differentiation of the cell type of interest is verified by analyzing cell type specific markers, gene expression profile, and functional assays. [1]

Early methods

support cells and matrices provide developmental-like environmental signals. [8]

Current methodologies

Directed differentiation

This method consists in exposing the cells to specific signaling pathways modulators and manipulating cell culture conditions (environmental or exogenous) to mimick the natural sequence of developmental decisions to produce a given cell type/tissue. [1] [8] A drawback of this approach is the necessity to have a good understanding of how the cell type of interest is formed. [1]

Direct reprogramming

This method, also known as transdifferentiation or direct conversion, consists in overexpressing one or several factors, usually transcription factors, introduced in the cells. [1] The starting material can be either pluripotent stem cells (PSCs), or either differentiated cell type such as fibroblasts. The principle was first demonstrated in 1987 with the myogenic factors MyoD. [9] A drawback of this approach is the introduction of foreign nucleic acid in the cells and the forced expression of transcription factors which effects are not fully understood.

Lineage/cell type-specific selection

This methods consists in selecting the cell type of interest, usually with antibiotic resistance. For this purpose, the source material cells are modified to contain antibiotic resistance cassette under a target cell type specific promoter. [10] [11] Only cells committed to the lineage of interest is surviving the selection.

Applications

Directed differentiation provides a potentially unlimited and manipulable source of cell and tissues. Some applications are impaired by the immature phenotype of the pluripotent stem cells (PSCs)-derived cell type, which limits the physiological and functional studies possible. [6] Several application domains emerged:

Model system for basic science

For basic science, notably developmental biology and cell biology, PSC-derived cells allow to study at the molecular and cellular levels fundamental questions in vitro, [5] that would have been otherwise extremely difficult or impossible to study for technical and ethical reasons in vivo such as embryonic development of human. In particular, differentiating cells are amenable for quantitative and qualitative studies. [8] More complex processes can also be studied in vitro and formation of organoids, including cerebroids, optic cup and kidney have been described.

Drug discovery and toxicology

Cell types differentiated from pluripotent stem cells (PSCs) are being evaluated as preclinical in vitro models of Human diseases. [5] Human cell types in a dish provide an alternative to traditional preclinical assays using animal, human immortalized cells or primary cultures from biopsies, which have their limitations. Clinically relevant cell types i.e. cell type affected in diseases are a major focus of research, this includes hepatocytes, Langerhans islet beta-cells, [12] cardiomyocytes and neurons. Drug screen are performed on miniaturized cell culture in multiwell-plates or on a chip. [6]

Disease modeling

PSCs-derived cells from patients are used in vitro to recreate specific pathologies. [6] The specific cell type affected in the pathology is at the base of the model. For example, motoneurons are used to study spinal muscular atrophy (SMA) and cardiomyocytes [2] are used to study arrythmia. This can allow for a better understanding of the pathogenesis and the development of new treatments through drug discovery. [6] Immature PSC-derived cell types can be matured in vitro by various strategies, such as in vitro ageing, to modelize age-related disease in vitro. Major diseases being modelized with PSCs-derived cells are amyotrophic lateral sclerosis (ALS), Alzheimer's (AD), Parkinson's (PD), fragile X syndrome (FXS), Huntington disease (HD), Down syndrome, Spinal muscular atrophy (SMA), muscular dystrophies, [13] [14] cystic fibrosis, Long QT syndrome, and Type I diabetes. [6]

Regenerative medicine

The potentially unlimited source of cell and tissues may have direct application for tissue engineering, cell replacement and transplantation following acute injuries and reconstructive surgery. [2] [5] These applications are limited to the cell types that can be differentiated efficiently and safely from human PSCs with the proper organogenesis. [1] Decellularized organs are also being used as tissue scaffold for organogenesis. Source material can be normal healthy cells from another donor (heterologous transplantation) or genetically corrected from the same patient (autologous). Concerns on patient safety have been raised due to the possibility of contaminating undifferentiated cells. The first clinical trial using hESC-derived cells was in 2011. [15] The first clinical trial using hiPSC-derived cells started in 2014 in Japan. [16]

See also

Related Research Articles

<span class="mw-page-title-main">Stem cell</span> Undifferentiated biological cells that can differentiate into specialized cells

In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can change into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of cell in a cell lineage. They are found in both embryonic and adult organisms, but they have slightly different properties in each. They are usually distinguished from progenitor cells, which cannot divide indefinitely, and precursor or blast cells, which are usually committed to differentiating into one cell type.

<span class="mw-page-title-main">Fibroblast</span> Animal connective tissue cell

A fibroblast is a type of biological cell typically with a spindle shape that synthesizes the extracellular matrix and collagen, produces the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of connective tissue in animals.

Transdifferentiation, also known as lineage reprogramming, is the process in which one mature somatic cell is transformed into another mature somatic cell without undergoing an intermediate pluripotent state or progenitor cell type. It is a type of metaplasia, which includes all cell fate switches, including the interconversion of stem cells. Current uses of transdifferentiation include disease modeling and drug discovery and in the future may include gene therapy and regenerative medicine. The term 'transdifferentiation' was originally coined by Selman and Kafatos in 1974 to describe a change in cell properties as cuticle producing cells became salt-secreting cells in silk moths undergoing metamorphosis.

<span class="mw-page-title-main">Cellular differentiation</span> Developmental biology

Cellular differentiation is the process in which a stem cell changes from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Some differentiation occurs in response to antigen exposure. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly controlled modifications in gene expression and are the study of epigenetics. With a few exceptions, cellular differentiation almost never involves a change in the DNA sequence itself. However, metabolic composition does get altered quite dramatically where stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. Thus, different cells can have very different physical characteristics despite having the same genome.

<span class="mw-page-title-main">Embryonic stem cell</span> Type of pluripotent blastocystic stem cell

Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells. Isolating the inner cell mass (embryoblast) using immunosurgery results in destruction of the blastocyst, a process which raises ethical issues, including whether or not embryos at the pre-implantation stage have the same moral considerations as embryos in the post-implantation stage of development.

Organogenesis is the phase of embryonic development that starts at the end of gastrulation and continues until birth. During organogenesis, the three germ layers formed from gastrulation form the internal organs of the organism.

<span class="mw-page-title-main">Embryoid body</span> Three-dimensional aggregate of pluripotent stem cells

Embryoid bodies (EBs) are three-dimensional aggregates formed by pluripotent stem cells. These include embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC)

<span class="mw-page-title-main">Oct-4</span> Mammalian protein found in Homo sapiens

Oct-4, also known as POU5F1, is a protein that in humans is encoded by the POU5F1 gene. Oct-4 is a homeodomain transcription factor of the POU family. It is critically involved in the self-renewal of undifferentiated embryonic stem cells. As such, it is frequently used as a marker for undifferentiated cells. Oct-4 expression must be closely regulated; too much or too little will cause differentiation of the cells.

<span class="mw-page-title-main">Adult stem cell</span> Multipotent stem cell in the adult body

Adult stem cells are undifferentiated cells, found throughout the body after development, that multiply by cell division to replenish dying cells and regenerate damaged tissues. Also known as somatic stem cells, they can be found in juvenile, adult animals, and humans, unlike embryonic stem cells.

<span class="mw-page-title-main">Stem-cell line</span> Culture of stem cells that can be propagated indefinitely

A stem cell line is a group of stem cells that is cultured in vitro and can be propagated indefinitely. Stem cell lines are derived from either animal or human tissues and come from one of three sources: embryonic stem cells, adult stem cells, or induced pluripotent stem cells. They are commonly used in research and regenerative medicine.

<span class="mw-page-title-main">Organoid</span> Miniaturized and simplified version of an organ

An organoid is a miniaturised and simplified version of an organ produced in vitro in three dimensions that mimics the key functional, structural, and biological complexity of that organ. It is derived from one or a few cells from a tissue, embryonic stem cells, or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities. The technique for growing organoids has rapidly improved since the early 2010s, and The Scientist named it one of the biggest scientific advancements of 2013. Scientists and engineers use organoids to study development and disease in the laboratory, for drug discovery and development in industry, personalized diagnostics and medicine, gene and cell therapies, tissue engineering, and regenerative medicine.

Neural stem cells (NSCs) are self-renewing, multipotent cells that firstly generate the radial glial progenitor cells that generate the neurons and glia of the nervous system of all animals during embryonic development. Some neural progenitor stem cells persist in highly restricted regions in the adult vertebrate brain and continue to produce neurons throughout life. Differences in the size of the central nervous system are among the most important distinctions between the species and thus mutations in the genes that regulate the size of the neural stem cell compartment are among the most important drivers of vertebrate evolution.

<span class="mw-page-title-main">Induced pluripotent stem cell</span> Pluripotent stem cell generated directly from a somatic cell

Induced pluripotent stem cells are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka and Kazutoshi Takahashi in Kyoto, Japan, who together showed in 2006 that the introduction of four specific genes, collectively known as Yamanaka factors, encoding transcription factors could convert somatic cells into pluripotent stem cells. Shinya Yamanaka was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent."

<span class="mw-page-title-main">Cell potency</span> Ability of a cell to differentiate into other cell types

Cell potency is a cell's ability to differentiate into other cell types. The more cell types a cell can differentiate into, the greater its potency. Potency is also described as the gene activation potential within a cell, which like a continuum, begins with totipotency to designate a cell with the most differentiation potential, pluripotency, multipotency, oligopotency, and finally unipotency.

Cellular cardiomyoplasty, or cell-based cardiac repair, is a new potential therapeutic modality in which progenitor cells are used to repair regions of damaged or necrotic myocardium. The ability of transplanted progenitor cells to improve function within the failing heart has been shown in experimental animal models and in some human clinical trials. In November 2011, a large group of collaborators at Minneapolis Heart Institute Foundation at Abbott Northwestern found no significant difference in left ventricular ejection fraction (LVEF) or other markers, between a group of patients treated with cellular cardiomyoplasty and a group of control patients. In this study, all patients were post MI, post percutaneous coronary intervention (PCI) and that infusion of progenitor cells occurred 2–3 weeks after intervention. In a study that is currently underway, however, more positive results were being reported: In the SCIPIO trial, patients treated with autologous cardiac stem cells post MI have been reported to be showing statistically significant increases in LVEF and reduction in infarct size over the control group at four months after implant. Positive results at the one-year mark are even more pronounced. Yet the SCIPIO trial "was recently called into question". Harvard University is "now investigating the integrity of some of the data". The Lancet recently published a non-specific ‘Expression of concern’ about the paper. Subsequently, another preclinical study also raised doubts on the rationale behind using this special kind of cell, as it was found that the special cells only have a minimal ability in generating new cardiomyocytes. Some specialists therefore now raise concerns to continue.

<span class="mw-page-title-main">P19 cell</span>

P19 cells is an embryonic carcinoma cell line derived from an embryo-derived teratocarcinoma in mice. The cell line is pluripotent and can differentiate into cell types of all three germ layers. Also, it is the most characterized embryonic carcinoma (EC) cell line that can be induced into cardiac muscle cells and neuronal cells by different specific treatments. Indeed, exposing aggregated P19 cells to dimethyl sulfoxide (DMSO) induces differentiation into cardiac and skeletal muscle. Also, exposing P19 cells to retinoic acid (RA) can differentiate them into neuronal cells.

Human engineered cardiac tissues (hECTs) are derived by experimental manipulation of pluripotent stem cells, such as human embryonic stem cells (hESCs) and, more recently, human induced pluripotent stem cells (hiPSCs) to differentiate into human cardiomyocytes. Interest in these bioengineered cardiac tissues has risen due to their potential use in cardiovascular research and clinical therapies. These tissues provide a unique in vitro model to study cardiac physiology with a species-specific advantage over cultured animal cells in experimental studies. hECTs also have therapeutic potential for in vivo regeneration of heart muscle. hECTs provide a valuable resource to reproduce the normal development of human heart tissue, understand the development of human cardiovascular disease (CVD), and may lead to engineered tissue-based therapies for CVD patients.

Gordon M. Keller is a Canadian scientist recognized for his research on applying developmental biology findings to in vitro pluripotent stem cell differentiation. He is currently a Senior Scientist at the Ontario Cancer Institute, a Professor at the University of Toronto and the director of the McEwen Centre for Regenerative Medicine.

<span class="mw-page-title-main">Christine L. Mummery</span>

Christine L. Mummery (1953) is an appointed professor of Developmental Biology at Leiden University and the head of the Department of Anatomy and Embryology at Leiden University Medical Center in the Netherlands.

A myelinoid or myelin organoid is a three dimensional in vitro cultured model derived from human pluripotent stem cells (hPSCs) that represents various brain regions, the spinal cord or the peripheral nervous system in early fetal human development. Myelinoids have the capacity to recapitulate aspects of brain developmental processes, microenvironments, cell to cell interaction, structural organization and cellular composition. The differentiating aspect dictating whether an organoid is deemed a cerebral organoid/brain organoid or myelinoid is the presence of myelination and compact myelin formation that is a defining feature of myelinoids. Due to the complex nature of the human brain, there is a need for model systems which can closely mimic complicated biological processes. Myelinoids provide a unique in vitro model through which myelin pathology, neurodegenerative diseases, developmental processes and therapeutic screening can be accomplished.

References

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