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 o ...

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, asexual reproduction, metamorphosis, and the growth and differentiation of ste ...

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 biologi ...

. 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 cells ...

or tissue of interest.

Stem cells In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can differentiate into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of ...

are by definition

pluripotent Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta. According to Latin terms, Pluripotentia means the ability for many thin ...

, able to differentiate into several cell types such as

neurons A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. ...

cardiomyocytes Cardiac muscle (also called heart muscle, myocardium, cardiomyocytes and cardiac myocytes) is one of three types of vertebrate muscle tissues, with the other two being skeletal muscle and smooth muscle. It is an involuntary, striated muscle tha ...

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, bi ...

, 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 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 two or three pr ...

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 Emb ...

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 gast ...

) 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 the branch of mathematics concerning numerical descriptions of how likely an event is to occur, or how likely it is that a proposition is true. The probability of an event is a number between 0 and 1, where, roughly speaking, ...

stochastic Stochastic (, ) refers to the property of being well described by a random probability distribution. Although stochasticity and randomness are distinct in that the former refers to a modeling approach and the latter refers to phenomena themselve ...

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, m ...

analysis, lineage tracing, embryo micro-manipulation and

gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. ...

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 ...

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 experimental medicine, encompasses a wide array of research, extending from " basic research" (also called ''bench science'' or ''bench research''), – involving fundamental scienti ...

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 In biology, the extracellular matrix (ECM), also called intercellular matrix, is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide struct ...

promoting cell adhesion and differentiation, and define

culture media A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss ''Physcomitrella patens''. Differe ...

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 re ...

small molecules Within the fields of 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 ...

, 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 cell A fibroblast is a type of biological cell 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 ...

s, 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 Fetal bovine serum (FBS) is derived from the blood drawn from a bovine fetus via a closed system of collection at the slaughterhouse. Fetal bovine serum is the most widely used serum-supplement for the in vitro cell culture of eukaryotic cells. Th ...

, 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. It is a typ ...

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) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistanc ...

. 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 or fundamental research, is a type of scientific research with the aim of improving scientific theories for better understanding and prediction of natural or other phenomena. In contrast, applied resea ...

, 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 a ...

, 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 The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; bloo ...

have been described.

Drug discovery and toxicology

Cell types differentiated from pluripotent stem cells (PSCs) are being evaluated as

preclinical In drug development, preclinical development, also termed preclinical studies or nonclinical studies, is a stage of research that begins before clinical trials (testing in humans) and during which important feasibility, iterative testing and drug ...

in vitro models of Human diseases. Human cell types in a dish provide an alternative to traditional preclinical assays using animal, human immortalized cells or primary cultures from

biopsies A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist. The process involves extraction of sample cells or tissues for examination to determine the presence or extent of a di ...

, 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 Beta cells (β-cells) are a type of cell found in pancreatic islets that synthesize and secrete insulin and amylin. Beta cells make up 50–70% of the cells in human islets. In patients with Type 1 diabetes, beta-cell mass and function are dimi ...

and

Drug screen A drug is any chemical substance that causes a change in an organism's physiology or psychology when consumed. Drugs are typically distinguished from food and substances that provide nutritional support. Consumption of drugs can be via inhalati ...

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 genet ...

(SMA) and cardiomyocytes are used to study

arrythmia Arrhythmias, also known as cardiac arrhythmias, heart arrhythmias, or dysrhythmias, are irregularities in the heartbeat, including when it is too fast or too slow. A resting heart rate that is too fast – above 100 beats per minute in adults ...

. This can allow for a better understanding of the

pathogenesis 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 from Greek πάθος ''pat ...

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 IN, In or in may refer to: Places * India (country code IN) * Indiana, United States (postal code IN) * Ingolstadt, Germany (license plate code IN) * In, Russia, a town in the Jewish Autonomous Oblast Businesses and organizations * In ...

, to modelize 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 Lou Gehrig's disease, is a neurodegenerative disease that results in the progressive loss of motor neurons that control voluntary muscles. ALS is the most comm ...

(ALS),

Alzheimer's Alzheimer's disease (AD) is a neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As ...

(AD),

Parkinson's Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms become ...

(PD),

fragile X syndrome Fragile X syndrome (FXS) is a genetic disorder characterized by mild-to-moderate intellectual disability. The average IQ in males with FXS is under 55, while about two thirds of affected females are intellectually disabled. Physical features may ...

(FXS),

Huntington disease Huntington's disease (HD), also known as Huntington's chorea, is a neurodegenerative disease that is mostly inherited. The earliest symptoms are often subtle problems with mood or mental abilities. A general lack of coordination and an unst ...

(HD),

Down syndrome Down syndrome or Down's syndrome, also known as trisomy 21, is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. It is usually associated with child development, physical growth delays, mild to moderate ...

, 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 affe ...

cystic fibrosis Cystic fibrosis (CF) is a rare genetic disorder that affects mostly the lungs, but also the pancreas, liver, kidneys, and intestine. Long-term issues include difficulty breathing and coughing up mucus as a result of frequent lung infections. ...

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 fainting, ...

, and

Type I diabetes Type 1 diabetes (T1D), formerly known as juvenile diabetes, is an autoimmune disease that originates when cells that make insulin (beta cells) are destroyed by the immune system. Insulin is a hormone required for the cells to use blood sugar for ...

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 implica ...

. 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

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