Directed differentiation is a

bioengineering Biological engineering or bioengineering is the application of principles of biology and the tools of engineering to create usable, tangible, economically viable products. Biological engineering employs knowledge and expertise from a number ...

methodology In its most common sense, methodology is the study of research methods. However, the term can also refer to the methods themselves or to the philosophical discussion of associated background assumptions. A method is a structured procedure for bri ...

at the interface of stem cell biology,

developmental biology Developmental biology is the study of the process by which animals and plants grow and develop. Developmental biology also encompasses the biology of Regeneration (biology), regeneration, asexual reproduction, metamorphosis, and the growth and di ...

and

tissue engineering Tissue engineering is a biomedical engineering discipline that uses a combination of cells, engineering, materials methods, and suitable biochemical and physicochemical factors to restore, maintain, improve, or replace different types of biolo ...

. It is essentially harnessing the potential of stem cells by constraining their differentiation in vitro toward a specific

cell type A cell type is a classification used to identify cells that share morphological or phenotypical features. A multicellular organism may contain cells of a number of widely differing and specialized cell types, such as muscle cells and skin cell ...

or tissue of interest.

Stem 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 ...

are by definition

pluripotent 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 ...

, able to differentiate into several cell types such as

neurons A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...

, cardiomyocytes,

hepatocytes A hepatocyte is a cell of the main parenchymal tissue of the liver. Hepatocytes make up 80% of the liver's mass. These cells are involved in: * Protein synthesis * Protein storage * Transformation of carbohydrates * Synthesis of cholesterol, bile ...

, etc. Efficient ''directed differentiation'' requires a detailed understanding of the lineage and cell fate decision, often provided by developmental biology.

Conceptual frame

During differentiation,

cells make a number of developmental decisions to generate first the three

germ layer A germ layer is a primary layer of cell (biology), cells that forms during embryonic development. The three germ layers in vertebrates are particularly pronounced; however, all eumetazoans (animals that are sister taxa to the sponges) produce tw ...

s (

ectoderm The ectoderm is one of the three primary germ layers formed in early embryonic development. It is the outermost layer, and is superficial to the mesoderm (the middle layer) and endoderm (the innermost layer). It emerges and originates from the o ...

mesoderm The mesoderm is the middle layer of the three germ layers that develops during gastrulation in the very early development of the embryo of most animals. The outer layer is the ectoderm, and the inner layer is the endoderm.Langman's Medical ...

and

endoderm Endoderm is the innermost of the three primary germ layers in the very early embryo. The other two layers are the ectoderm (outside layer) and mesoderm (middle layer). Cells migrating inward along the archenteron form the inner layer of the gastr ...

) of the embryo and intermediate progenitors, followed by subsequent decisions or check points, giving rise to all the body's mature tissues. The differentiation process can be modeled as sequence of binary decisions based on

probabilistic Probability is a branch of mathematics and statistics concerning events and numerical descriptions of how likely they are to occur. The probability of an event is a number between 0 and 1; the larger the probability, the more likely an e ...

stochastic Stochastic (; ) is the property of being well-described by a random probability distribution. ''Stochasticity'' and ''randomness'' are technically distinct concepts: the former refers to a modeling approach, while the latter describes phenomena; i ...

models. Developmental biology and embryology provides the basic knowledge of the cell types' differentiation through

mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, ...

analysis, lineage tracing, embryo micro-manipulation and

gene expression Gene expression is the process (including its Regulation of gene expression, regulation) by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, ...

studies. Cell differentiation and tissue

organogenesis 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 (the ectoderm, endoderm, and mesoderm) form the internal org ...

involve a limited set of developmental signaling pathways. 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 Medical research (or biomedical research), also known as health research, refers to the process of using scientific methods with the aim to produce knowledge about human diseases, the prevention and treatment of illness, and the promotion of ...

applications. Since the discovery of embryonic stem (ES) cells (1981) and induced pluripotent stem (iPS) cells (2006), source material is potentially unlimited. Historically, embryonic carcinoma (EC) cells have also been used. Fibroblasts or other differentiated cell types have been used for direct reprogramming strategies.

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. 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. A limited number of signaling factors such as

growth factors A growth factor is a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. Usually it is a secreted protein or a steroid hormone. Growth factors are important for regu ...

small molecules In molecular biology and pharmacology, a small molecule or micromolecule is a low molecular weight (≤ 1000 daltons) organic compound that may regulate a biological process, with a size on the order of 1 nm. Many drugs are small molecules; t ...

, controlling cell differentiation, is applied sequentially or in a combinatorial manner, at varying dosage and exposure time. Proper differentiation of the cell type of interest is verified by analyzing cell type specific markers,

profile, and functional assays.

Early methods

* co-culture with stromal cells or feeder cells, and on specific culture substrates: support cells and matrices provide developmental-like environmental signals. * 3D cell aggregate formation, termed embryoid bodies (EBs): the aggregate aim at mimicking early embryonic development and instructing the cell differentiation. * culture in presence of fetal bovine serum, removal of pluripotency factors.

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. A drawback of this approach is the necessity to have a good understanding of how the cell type of interest is formed.

Direct reprogramming

This method, also known as

transdifferentiation 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.(a process whe ...

or direct conversion, consists in overexpressing one or several factors, usually transcription factors, introduced in the cells. 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. 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 Antimicrobial resistance (AMR or AR) occurs when microbes evolve mechanisms that protect them from antimicrobials, which are drugs used to treat infections. This resistance affects all classes of microbes, including bacteria (antibiotic resis ...

. For this purpose, the source material cells are modified to contain antibiotic resistance cassette under a target cell type specific promoter. Only cells committed to the lineage of interest is surviving the

selection Selection may refer to: Science * Selection (biology), also called natural selection, selection in evolution ** Sex selection, in genetics ** Mate selection, in mating ** Sexual selection in humans, in human sexuality ** Human mating strat ...

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. Several application domains emerged:

Model system for basic science

For

basic science Basic research, also called pure research, fundamental research, basic science, or pure science, is a type of scientific research with the aim of improving scientific theories for better understanding and prediction of natural or other phenomen ...

, notably

and

cell biology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living an ...

, PSC-derived cells allow to study at the molecular and cellular levels fundamental questions in vitro, 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. More complex processes can also be studied in vitro and formation of organoids, including cerebroids, optic cup and

kidney In humans, the kidneys are two reddish-brown bean-shaped blood-filtering organ (anatomy), organs that are a multilobar, multipapillary form of mammalian kidneys, usually without signs of external lobulation. They are located on the left and rig ...

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. Human cell types in a dish provide an alternative to traditional preclinical assays using animal, human

immortalized cells Biological immortality (sometimes referred to as bio-indefinite mortality) is a state in which the rate of mortality from senescence (or aging) is stable or decreasing, thus decoupling it from chronological age. Various unicellular and multicellul ...

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

, Langerhans islet beta-cells, cardiomyocytes and

. Drug screen are performed on miniaturized cell culture in multiwell-plates or on a chip.

Disease modeling

PSCs-derived cells from patients are used in vitro to recreate specific pathologies. 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 Spinal muscular atrophy (SMA) is a rare neuromuscular disorder that results in the loss of motor neurons and progressive muscle wasting. It is usually diagnosed in infancy or early childhood and if left untreated it is the most common geneti ...

(SMA) and cardiomyocytes are used to study

arrhythmia Arrhythmias, also known as cardiac arrhythmias, are irregularities in the cardiac cycle, heartbeat, including when it is too fast or too slow. Essentially, this is anything but normal sinus rhythm. A resting heart rate that is too fast – ab ...

. This can allow for a better understanding of the

pathogenesis In pathology, pathogenesis is the process by which a disease or disorder develops. It can include factors which contribute not only to the onset of the disease or disorder, but also to its progression and maintenance. The word comes . Descript ...

and the development of new treatments through drug discovery. Immature PSC-derived cell types can be matured in vitro by various strategies, such as in vitro ageing, to model age-related disease in vitro. Major diseases being modelized with PSCs-derived cells are

amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND) or—in the United States—Lou Gehrig's disease (LGD), is a rare, Terminal illness, terminal neurodegenerative disease, neurodegenerative disorder that results i ...

(ALS),

Alzheimer's Alzheimer's disease (AD) is a neurodegenerative disease and the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As the disease advances, symptoms can include problems wit ...

(AD),

Parkinson's Parkinson's disease (PD), or simply Parkinson's, is a neurodegenerative disease primarily of the central nervous system, affecting both motor and non-motor systems. Symptoms typically develop gradually and non-motor issues become more prevalen ...

(PD),

fragile X syndrome Fragile X syndrome (FXS) is a genetic neurodevelopmental disorder. The average IQ in males with FXS is under 55, while affected females tend to be in the borderline to normal range, typically around 70–85. Physical features may include a lo ...

(FXS), Huntington disease (HD), Down syndrome, Spinal muscular atrophy (SMA),

muscular dystrophies Muscular dystrophies (MD) are a genetically and clinically heterogeneous group of rare neuromuscular diseases that cause progressive weakness and breakdown of skeletal muscles over time. The disorders differ as to which muscles are primarily aff ...

cystic fibrosis Cystic fibrosis (CF) is a genetic disorder inherited in an autosomal recessive manner that impairs the normal clearance of Sputum, mucus from the lungs, which facilitates the colonization and infection of the lungs by bacteria, notably ''Staphy ...

Long QT syndrome Long QT syndrome (LQTS) is a condition affecting repolarization (relaxing) of the heart after a heartbeat, giving rise to an abnormally lengthy QT interval. It results in an increased risk of an irregular heartbeat which can result in fainti ...

, and Type I diabetes.

Regenerative medicine

The potentially unlimited source of cell and tissues may have direct application for

, cell replacement and transplantation following acute injuries and

reconstructive surgery Reconstructive surgery is surgery performed to restore normal appearance and function to body parts malformed by a disease or medical condition. Description Reconstructive surgery is a term with training, clinical, and reimbursement implicat ...

. These applications are limited to the cell types that can be differentiated efficiently and safely from human PSCs with the proper

. 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. The first clinical trial using hiPSC-derived cells started in 2014 in Japan.

References

{{Portal bar, Biology, Medicine Developmental biology Induced stem cells Stem cells Biotechnology Cell biology Biological engineering Biomedical engineering Tissue engineering