Stem cells: An insight into the therapeutic aspects from medical and dental perspectives

Abstract

The recent advancements in the field of stem cell (SC) biology have increased the hope of achieving the definitive treatments for the diseases which are now considered incurable such as diabetes, Parkinson's disease and other chronic long standing conditions. To achieve this possibility, it is necessary to understand the basic concepts of SC biology to utilize in various advanced techniques of regenerative medicine including tissue engineering and gene therapy. This article highlights the types of SCs available and their therapeutic capacity in regenerative medical and dental fields.

A stem cell (SC) is a cell which has an immense capacity for self-renewal and potency. SCs are found in all multicellular organisms, and they are characterized by the ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cell types.[1]

Stem cells can generate daughter cells identical to their mother (self-renewal) as well as produce progeny with more restricted potential (differentiated cells).[2] By definition, “a SC is capable of self-renewal, differentiation into at least one cell type and functional reconstitution of the tissue of origin.”[3]

This self-renewal capacity underlines the ability of adult SCs such as spermatogonial SCs, and hematopoietic SCs (HSCs) to constantly renew tissues that turn over rapidly and also where cells do not turn over so rapidly in the adult brain. There are long-lived quiescent SCs that may be reactivated to repair damage.[4]

Much current research is focused on the SCs identification, their characterization and isolation from the adult, harboring the hope that such cells may be useful for therapeutic repair of adult tissues either by exogenous cell therapy or by reactivation of endogenous SCs. Biologists have explored the development of embryos of all aspects, from worms to humans, in search of the answer to the question of how a single cell, the fertilized egg forms a complex organism. Cells are initially proliferative and pluripotent during embryogenesis; they only gradually become restricted to different cell fates.[5]

During embryogenesis, SCs can differentiate into all of the specialized embryonic tissues whereas in adult organisms, progenitor cells as well as SCs act as a repair system for the body; replenishing specialized cells while also maintaining the normal turnover of regenerative organs such as blood, skin, or intestinal tissues.[6,7,8]

Commonly, SCs come from two main sources:

• Embryos formed during the blastocyst phase of embryological development (embryonic stem cells [ESCs]) and

• Adult tissues (adult SCs).

Stem cells can now be grown and transformed into specialized cells with characteristics consistent with cells of various tissues such as muscles or nerves through cell culture. Highly plastic adult SCs derived from a variety of sources, such as bone marrow and umbilical cord blood; are routinely used in medical treatments. Embryonic cell lines and autologous ESCs generated through therapeutic cloning have also been proposed as promising candidates for future therapies.[9]

Earlier, it was thought that only ESCs can give rise to the complete range of cells in the organism but recent studies have revealed that adult SCs are unexpectedly common and indicate that they might be more plastic in their ability to differentiate into cell types of all the three germ layers than previously appreciated.[9,10] The presence of multipotent SCs in the adult might open up new therapeutic opportunities on the basis of tissue and organ replacement.[10]

With the latest advancement in research the SCs of a particular tissue is defined as: (a) Undifferentiated cells (b) capable of proliferation, (c) able to self-maintain the population, (d) capable of producing a large number of differentiated, functional progeny, (e) capable of regenerating the injured tissues, and (f) flexible use of these options.[10,11,12,13,14,15]

Properties of Stem Cells

Stem cells differ from other kinds of cells in the body. Regardless of their source, all SCs have three general properties:[3,16,17,18]

• They are unspecialized

• They are capable of dividing and self-renewing themselves for long periods; and

• They can give rise to specialized cell types.

Stem cells are unspecialized (relativity)

One of the fundamental properties of a SC is that it does not have any tissue-specific structures that allows it to perform specialized functions like pumping blood through the body, carrying molecules of oxygen through the bloodstream but they can give rise to specialized cells, including heart muscle cells or blood cells.[3,16,17,18]

Stem cells are capable of dividing and self-renewing themselves for long periods (self-maintenance and renewal)

Unlike the fully differentiated cells (nerve cells, blood cells, or muscle cells) which do not normally replicate themselves, SCs are capable of replicating many times. Replication of cells themselves multiple times without differentiation is called proliferation.[18]

Stem cells can give rise to specialized cells (potency)

When unspecialized SCs give rise to specialized cells, the process is called differentiation. Differentiation may be controlled by either internal signals (cell's genes) or external signals (chemicals secreted by other cells, certain cytokines molecules in the microenvironment and physical contact with the adjacent cells).[17,18]

Previously, it was considered that adult SCs typically generate the cell types of the tissue in which they reside. However, recent experiments over the last several years have proved that SCs from one tissue may be able to give rise to cell types of a completely different tissue, and this is known as plasticity. Blood cells differentiating into neurons and HSCs that can develop into heart muscles are a few examples of plasticity. Therefore, exploring the possibility of using adult SCs for cell-based therapies has become very active now-a-day.[17,18]

Classification of Stem Cells

Stem cells can be classified in the following ways: (1) According to their potency, (2) according to their origin, and/or (3) according to their origin, differentiation potency, and progeny.

Classification according to their potency

Stem cells are categorized by their potential to differentiate into other types of cells. The full classification includes.

Totipotent

Totipotency is the ability of a single cell to divide and produce all the differentiated cells including extraembryonic tissues of an organism. Spores and zygotes are examples for totipotent cells. Human development begins with a sperm fertilizes an egg to produce a single totipotent cell (zygote) which in turn divides into identical totipotent cells in the first hours after fertilization.[19,20,21]

Pluripotent

Pluripotent SC that has the potential to differentiate into any of the three germ layers: Ectoderm, endoderm or mesoderm. E.g., ESCs isolated from inner cell mass (ICM) of blastocysts. Any fetal or adult cell types can be derived from pluripotent SCs, but they lack the ability to contribute to extraembryonic tissue like the placenta.[19,20,21]

Multipotent

Multipotent progenitor cells have the potential to give rise to cells from multiple, but a limited number of lineages. E.g., HSCs - A blood SC that can develop into several types of blood cells, but lack the potential to develop into brain cells and other types of cells. Scientists have long held the opinion that differentiated cells cannot be altered or induced to behave in any way other than the way in which they have been naturally committed. In recent SC experiments, scientists have been able to persuade blood SCs to behave like neurons or brain cells - A process known as transdifferentiation.[19,20,21]

Oligopotent

An oligopotent cell has the potency to differentiate into a few cell types. Examples of progenitor cells are vascular SCs which have the capacity to become either endothelial or smooth muscle cells.[19,20,21]

Unipotent

A unipotent cell or precursor cell is one that has the capacity to differentiate into only one type of cell/tissue type. The most common example of these cells in humans is skin cells. This cell has a unique property: Self-renewal.[19,20,21]

Classification according to the origin

• Human ESCs (hESCs)[22,23]

• Fetal SCs[24]

• Infant SCs[25]

• Adult SCs.[26,27,28,29,30,31,32,33,34]

Human embryonic stem cells

The ICM of the 5–6-day old human blastocyst is the source of pluripotent ESCs. The ICM is composed of 30–34 cells. During embryonic development, the ICM develops into two distinct cell layers, the epiblast and hypoblast. The hypoblast forms the yolk sac which later becomes redundant in the human and the epiblast differentiates into the three primordial germ layers (ectoderm, mesoderm, and endoderm). Human embryonic germ cells which are also SCs, develop from the primordial germ cells of the gonadal ridge of 5–9-week old fetuses. These SCs are pluripotent and are able to produce cells of all three germ layers.[22,23]

Fetal stem cells

Fetal SCs are primitive cell types found in the organs of fetuses. Neural crest SCs, fetal HSCs, and pancreatic islet progenitors have been isolated in abortuses.[24]

Infant stem cells

Infant SCs are isolated from umbilical cord blood and Wharton's jelly. Umbilical cord blood contains circulating SCs, and the cellular contents of umbilical cord blood appear to be quite distinct from those of the bone marrow and adult peripheral blood. Cord blood SCs have been shown to be multipotent by being able to differentiate into neurons and liver cells. Matrix cells from the umbilical cord contain potentially useful SCs. This matrix termed as Wharton's jelly, has been a source for isolation of mesenchymal SCs (MSCs).[25]

Adult stem cells

1. Hematopoietic SCs (bone marrow and peripheral blood)

   Bone marrow possesses SCs that are both hematopoietic and mesenchymal in origin. The HSC is derived early in embryogenesis from the mesoderm and becomes deposited in very specific hematopoietic sites within the embryo. These sites include the bone marrow, liver, and yolk sac.[26,29,31]

2. MSCs (bone marrow stroma)

   Mesenchymal SCs are found postnatally in the nonhematopoietic bone marrow stroma. MSCs are multipotent cells that are capable of differentiating into cartilage, bone, muscle, tendon, ligament and fat tissues.[27,32,34]

3. Bone and cartilage SCs

4. Epidermal SCs (skin and hair)

   The epidermis harbors SCs at the base of the hair follicle and in the basal layer of the epidermis.[31,32]

5. Neuronal SCs

   The subventricular zone of the forebrain and the central gyrus of the hippocampus which are considered reservoirs of new neural cells.[27,28]

6. Pancreatic SCs

   Human pancreatic islets contained an unrecognized population of cells that expressed the neural SC (NSC)-specific marker nestin.[19,24]

7. Eye SCs (pigmented ciliary margin cells).[27,33]

   Classification according to their origin, differentiation potency and progeny[35] [Table 1].

Table 1.

Classification of stem cells

Sources of Tissue-Specific Stem Cells

(1) Endodermal origin (pulmonary epithelial SCs, gastrointestinal tract SCs, pancreatic SCs, hepatic oval cells, mammary and prostate gland SCs, ovarian and testicular SCs), (2) Mesodermal origin (hematopoietic SCs, mesenchymal stromal SC, cardiac SC, and satellite cells of muscles), (3) Ectodermal origin (NSCs, skin SCs, and ocular SCs).[31]

Basic Biology

Early mammalian embryogenesis is characterized by a gradual restriction in the developmental potential of the cells that constitute the embryo. The zygote and single blastomeres from a two to four cell embryo are totipotent. As the embryo continues to cleave, the blastomeres lose the potential to differentiate into all lineages. The blastocyst is the first landmark of the embryo in which lineage restriction is apparent. At this stage, the outer cells of the embryo compact into the trophectoderm, from which the placenta will derive. The inner cells, termed ICM, will give rise to all cell lineages of the embryo proper, but cannot contribute to the trophectoderm, and thus are considered pluripotent. As the embryo develops, the potency of the cells are more restricted, and they become multipotent, oligopotent, and unipotent.[36,37,38]

The four areas mainly concentrated on understanding the molecular mechanisms of ESC potency are: (1) Influence of interleukin-6 family cytokines, (2) extrinsic determinants of pluripotency, (3) intrinsic determinants of pluripotency and (4) epigenetic configurations. Cytokines and extrinsic determinants like leukemia inhibitory factor and other cytokines, ERK-MAPK pathway, fibroblast growth factor-4 (FGF-4), Wnt signaling pathway, transforming growth factor-β (TGF-β) and BMP, activin, and PI3/AKT pathway control the ESCs by up or down-regulating the potency and self-renewing properties.[39,40,41,42,43] Intrinsic determinants like transcription factors OCT-4, SOX-2, FOX-D3 and NANOG, and epigenetic mechanisms like polycomb-related complex-2, RNA interference system, nucleosome remodeling and histone deacetylation complex, and epigenetic modifiers like Ronin play a vital role in the self-renewal of ESCs. Likewise, by understanding the mechanism revolving around all types of SCs (fetal, infant and adult), it is possible to generate SCs with desired therapeutic properties.[36,44,45]

Applications of Stem Cells

The tremendous advancement in genetic, molecular and SC biology has led to the use of SCs in almost all the aspects of medical and dental fields to treat cancer, neurodegenerative diseases, heart diseases, muscular dystrophies, diabetes, burns, and skin ulcers, and applied in orthopedic and bone marrow transplantation treatments. The rapidly evolving techniques like tissue engineering and gene therapy use the SC concept as their basis to produce tissue organs for organ replacement and gene modification treatments.

Cancer Stem Cells: A Therapeutic Target in Cancer Therapy

The cancer SC (CSC) hypothesis has postulated that each tumor contains a subset of cells – the CSCs – that are uniquely responsible for tumor growth, heterogeneity, and metastasis. These specialized cells possess two key features that together distinguish them from the remainder of the tumor cells: Self-renewal and differentiation potential. The finding that leukemic SCs in acute myeloid leukemia share the same immunophenotype with normal HSCs, CD341, CD382, might suggest that cancers result from the oncogenic transformation of normal SCs. Similarly, observations about the time from carcinogenic exposure to the development of cancer also seem to support the notion that cancers are derived from normal tissue SCs.[46,47,48,49]

Traditionally, the only cells in a tissue thought to persist long enough to accumulate several oncogenic mutations and exhibit such a delayed effect, were the tissue SCs. Due to their rapid turnover, non-SCs that acquire mutations would be expected to die off before enough mutations could accumulate to transform them into cancer cells.[46,47,48,49]

The cancer stem cell model

At least two different models attempt to explain both clonality and tumor heterogeneity: The stochastic model; and the CSC model. The stochastic model is based on the notion that the transforming events that endow the cell of origin with a malignant phenotype cause all progeny of that cell also to be tumorigenic. In this model, tumor heterogeneity is caused by subclones of tumor cells that result from a combination of different microenvironments and random genetic changes (including point mutations, chromosomal rearrangements, aneuploidy, and gene amplifications). Ultimately, this model implies that effective treatment for cancer must involve eradication of all cancer cells.[46,47,48,49]

The CSC model postulates that only some tumor cells are tumorigenic - The CSC. When they divide, these malignant cells self-renew and also give rise to the non-self-renewing cells that go on to elaborate the heterogeneous cells within a cancer. In this way, CSCs drive tumor growth.[50]

Cancer stem cells and their clinical implications

The CSC model has many important clinical implications which might not only contribute a novel pathophysiological mechanism to explain tumors’ resistance to treatment, but it might also improve clinicians’ ability to more effectively diagnose, prognose, and treat cancer.[46,47,48,49]

Since the CSC model suggests that CSCs are responsible for the sustained and uncontrolled growth of malignancies, treatment failures in the clinic may be due partly to the resistance of CSCs to therapy. If CSCs are intrinsically more resistant to treatment (such as chemotherapy or radiotherapy) than other cells in the tumor, then treatment should increase the percentage of CSCs relative to pretreatment levels. Conversely, if all cells are similarly sensitive to treatment, then the relative percentages of tumorigenic and nontumorigenic cells should not change as a tumor shrinks which helps us to assess the prognosis.[46,47,48,49]

Central Nervous System Repair and Neural Stem Cells

The poor regenerative ability, particularly in the adult central nervous system, may be because of the limited number and restricted location of native NSCs, and/or limitations imposed by the surrounding microenvironment, which may not be supportive or instructive for neuronal differentiation for the most devastating of injuries is inadequate or ineffective. NSCs expanded ex vivo in culture, and then implanted into regions needing repair, may overcome those limitations related simply to inadequate numbers of NSCs near the defective region.[51] However, several transplantation experiments have suggested that neurogenic cues are transiently elaborated during degenerative processes (perhaps recapitulating developmental cues) and that exogenous NSCs are able to sense, home in on and respond appropriately to those cues.[52,53]

These disorders include rodent models of genetic and acquired (e.g., traumatic and ischemic) neurodegeneration, inheritable metabolic disorders, age-related degeneration, and neoplasms. NSCs differentiate robustly into neural cells, integrate flawlessly into neural parenchyma as multiple neural cell types (both neuronal and glial), respond to normal developmental and regeneration cues and migrate (even long distances) to multiple, disseminated areas of neuropathology. NSCs appear to be ideally suited for the molecular and cellular therapies required by extensive, diffuse degenerative processes. Examples of such widespread neurodegenerative conditions include myelin disorders, motor neuron degeneration, storage diseases, dementia conditions such as Alzheimer's disease and ischemic and traumatic pathologies like stroke.[54]

Use of ESCs for neural transplantation is in its formative stage, and only a limited amount of work has been completed with the spinal cord. Overall, the studies demonstrate that ESCs have a remarkable ability to integrate into the injured region of the cord and differentiate appropriately.[54]

Heart Disease and Stem Cells

It is now well-established that cardiomyocytes can be stably transplanted into normal or injured adult hearts. Cardiomyocytes derived from embryonic and fetal SCs or bone marrow and MSCs are used as donor cells to restore lost myocardial function to enhance angiogenesis and to provide support to the tissues.[55,56]

Moreover, transplanted donor cells can form a functional syncytium with the host myocardium and also it has been proved that the cardiomyocyte cells transplanted heart showed improvement in cardiac functions.[55,57,58] It has been contented that it is possible to provide large amount of human cardiomyocytes to fulfill the requirement of cells for transplantation and also favor cardiac regeneration with expected cardiac function.[59,60,61,62]

Muscular Dystrophy and Stem Cells

Muscular dystrophies are caused by progressive degeneration of skeletal muscle fibers. Sixty-five lack of one of the several proteins at the plasma membrane or within internal membranes, raises the probability of damage during contraction, and degeneration of fibers occur eventually. Fiber degeneration is counterbalanced by the regeneration of new fibers at the expense of resident myogenic cells, located underneath the basal lamina and known as satellite cells.[63,64] These cells should also produce new satellite cells to ensure a reserve population for further cycles of regeneration, and failure of this process results in the most severe forms of dystrophy.[65]

These other progenitors are probably derived from distinct anatomic sites, such as the microvascular niche of the skeletal muscle or bone marrow itself. In the most severe forms, like Duchenne's muscular dystrophy, regeneration is totally exhausted, and skeletal muscle is progressively replaced by fat and fibrous tissue. Subsequently, this condition leads the patient to progressive weakness and eventual death by respiratory failure, cardiac failure, or both.[63,64]

Current therapeutic approaches involve steroids and result in modest beneficial effects. Novel experimental approaches can be schematically grouped into three major areas.[66]

• The first is gene therapy aiming at the production of new viral vectors (mainly adeno-associated and lentiviral vectors, the latter for cell-mediated gene therapy) that are designed to be less antigenic and more efficient in transducing adult muscle fibers and/or myogenic cells[66]

• Novel pharmacological approaches focus on high-throughput screens for molecules that may interfere with pathogenic pathways. This aims to identify molecules that cause the skipping of termination codons or upregulate utrophin synthesis, a cognate protein that compensates for dystrophin absence when overexpressed in dystrophic mice; it also includes molecules that simply enhance muscle regeneration or delay protein degradation[67]

• The final group is cell therapy, based initially on myoblast transplantation, and more recently on the transplantation of stem progenitor cells.[68]

Diabetes and Stem Cells

The complications associated with diabetes (retinopathy, nephropathy, and neuropathy) require intensive treatments like pancreas transplantation or SC therapy which could provide long-term benefits to the patients. Pancreas transplantation performed since 1978 required major surgery and its associated complications heralded the development of new technique where transplant of only pancreatic islet cells to the liver through the portal vein via transhepatic angiography and it gained more attention since late 1980s. In general, poor outcomes were obtained throughout the 1990s, but the introduction of the Edmonton protocol in 2000 provided far better results, the improvement being due to better islet preparations, transplantation of more islets and improved immunosuppression.[69]

With the advancement in SC biology, SCs obtained from human ESCs cultured in gelatine or human fibroblast served its purpose better than Edmonton protocol method. Induced pluripotent SC is a recently developed SC and ethically has fewer controversies than hESCs and acts as a better source for the production of pancreatic islets for transplants. With the acquirement of knowledge on pdx-1, a transcription factor; it has been evidenced that adult SCs and precursor cells produced pancreatic islets apart from hESCs for the treatment of diabetes.[70]

Orthopedic Applications of Stem Cells

The inherent regenerative ability of the cartilage, tendon, and ligament is comparatively less than bone which has the tremendous regenerative capacity. Hence, concentrated use of SCs is required for repair of cartilage tendon and ligaments, where injuries to these tissues result in replacement of tissues with less organized scar tissues inferior in quality to the native tissue.[71] Chondral defects penetrating the subchondral bone usually do not require extensive treatment as it is repaired by the MSCs inherently present in the bone marrow, periosteum, and synovium.[71,72,73] And in areas where the defects are superficial and do not penetrate subchondral bone, lack of MSCs and vascularity for repair demands SC therapy using cultured MSCs.[71] Injuries to the ligaments and tendons may lead to inflammation or tear to these structures where the ultimate result would be degenerative joint diseases if not treated promptly. In normal healing process, healing is accelerated with the stimulation of fibroblasts by growth factors such as platelet-derived growth factor (PDGF), TGF-β, and FGF.[74] For example, ligaments like anterior collateral ligament show less tendency for repair compared to the medial collateral ligament and requires MSC gene therapy using viral vectors to deliver the growth factors to accelerate and organize the healing process.[75,76]

Burns and Skin Ulcers

Burns and skin ulcers are major causes of morbidity and, in the case of burns, mortality, in both the developed and developing world. The traditional methods to treat burns include autologous and allogenous skin grafting. Epidermal SCs which reside in the basal layer of epidermis are responsible for the ability of the epidermis to replace itself, both in normal circumstances and in traumatic conditions (burns and skin ulceration).[14,77]

In autologous skin grafting, epidermal cells have been used to treat burns since the introduction of the split skin graft by Karl Thiersh in the late 1800s. Skin grafting to cover defects caused by burns or skin ulceration is limited by the area of skin. Full-thickness grafts (including all of the epidermis and dermis) provide good cosmetic results but require a primary closure of the donor site, limiting the area that may be grafted.[78]

To overcome this problem, the use of split skin grafts was developed whereby epidermis and underlying dermis is shaved from the donor site to provide a graft and the donor site then re-epithelializes from the SCs present in the underlying hair follicles. The limitations of skin grafting techniques are the area that can be covered by them and the many weeks it may take to cover a large area of burn with autologous split skin. Burns of 80–90% are survivable in the short-term with resuscitation, but if the treatment is delayed because of a lack of grafts, results in a high morbidity and mortality rate.[79] To overcome these problems, Rheinwald and Green developed a technique for the serial cultivation of epidermal cells, producing a 1000-10,000 fold area of graftable epidermis than the initial biopsy. Then these epidermal sheets can then be grafted onto clean wound beds but they are sensitive to loss by bacterial infection and blistering. In full-thickness burns where the dermis has been lost, the cultured epidermal autograft may be placed directly onto muscle or fascia. These cultured epidermal autografts form a permanent covering, suggesting that the SCs initially cultured and then transplanted have been maintained as SCs and, therefore, retain their crucial role in epidermal maintenance.[80]

In allogenous skin grating technique, allografts were developed due to the lack of available donor sites for split-skin grafting in patients with massive burns as the time taken to grow cultured autologous skin from these patients is more. In order to prevent secondary sepsis and other complications associated with open burn wound, cadaveric skin is an alternative which is immediately available, plentiful, effective, and affordable. And it is always eventually rejected by the recipient as it is a true allograft. Alloderm is a processed human dermis from which the epidermal and dermal cells have been removed, leaving only the connective tissue matrix. Then alloderm can be applied to burns, and cultured autograft may be placed on top of it to prevent the early said complications. The currently available artificial dermis lacks a vascular plexus for the nourishment of the epidermis and requires host vasculogenesis into the dermis graft to supply nourishment to the grafted epidermis. Efforts have therefore been focused on encouraging the process of vasculogenesis by genetic engineering of grafts to produce growth factors and cytokines vital to this process.[81,82]

Skin ulcers

In ulcer therapy, the time constraints are not as severe and for definitive closure, split skin grafts remain the gold standard. Cultured skin has been used in the treatment of skin ulcers as a “living dressing” (particularly cultured allografts). Cultured, autologous outer root sheath cells used in the treatment of chronic decubitus ulcers have been found to produce an “edging effect” - The contraction of the chronic wounds edges in response to the graft, believed to be caused by a release of growth factors, cytokines, and hormones from the outer root sheath cells.[83] Apligraf is a cultured, bilayered living skin equivalent derived from neonatal foreskin keratinocytes, bovine type I collagen, and neonatal foreskin fibroblasts. It is indicated for the treatment of venous ulcers and neuropathic diabetic foot ulcers. Chronic wounds (e.g., those with dormant edges) re-epithelialize when exposed to living allograft material. This edge effect, like that seen with outer root sheath cells, is most probably caused by the presence of stimulatory factors. Chronic wounds heal better after repeated application of skin grafts. Hence, cultured autologous skin grafts offer better results, and suitable for larger area coverage and chronic ulcers.[84]

Tissue Engineering

Traditionally, approaches to restore tissue function have involved organ donation. However, the shortage of transplantable human tissues such as bone marrow, hearts, kidneys, livers, and pancreases required alternatives, where tissue engineering plays a vital role.[66,85] Tissue engineering-based therapies may provide a possible solution to alleviate the current shortage of organ donors. Biological and engineering principles are combined in tissue engineering techniques to produce cell-based substitutes. One of the major hurdles in engineering tissue constructs for clinical use is the limited availability of human cells as tissue source. SCs isolated from adults or developing embryos are a current source for cells for tissue engineering.[66,85]

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences to develop biological substitutes, usually composed of biological and synthetic components, which restore, maintain or improve tissue function. Tissue-engineered products would provide a life-long therapy which would reduce the hospitalization and healthcare costs associated with drug therapy while enhancing the patients’ quality of life. Tissue engineering uses any one of the following substitutes: (1) Isolated cells or cell substitutes as cellular replacement parts, (2) acellular materials capable of inducing tissue regeneration, (3) a combination of cells and materials (typically in the form of scaffolds).[86,87] Isolated cells have been used as a substitute for cell replacement parts for many years. In fact, the first application of SCs as a cellular replacement therapy is associated with bone marrow transplantation or blood transfusion studies, in which donor HSCs repopulated the host's blood cells.[88]

Tissue engineering approaches that use cells and scaffolds can be categorized into two categories; open and closed systems. In open tissue engineering systems, cells are immobilized within a highly-porous, three-dimensional scaffold. Scaffold could either be comprised of synthetic or natural materials or composites of both. Ideally, this scaffold provides the cells with a suitable growth environment, optimum oxygen and nutrient transport properties, good mechanical integrity, and a suitable degradation rate. The use of scaffolds provides three-dimensional environments and brings the cells in close proximity so that it provides the cells with sufficient time to enable self-assembly and formation of various components that are associated with the tissue microenvironment. Open tissue engineering systems have been successfully used to create a number of biological substitutes, like bone, cartilage, skin and tooth, etc.[71,89,90,91,92,93,94,95,96,97,98]

The closed systems aim to overcome this difficulty by immobilizing cells within polymeric matrices that provide a barrier for the immunological components of the host. The implants can either be implanted into the patient directly or used as extra-corpical devices. Closed tissue engineering systems have been used particularly for the treatment of diabetes, liver, and Parkinson's disease.[71,89,90,91,92,93,94,95,96,97,98]

Synthetic scaffolds that support tissue growth by serving as the extracellular matrix for the cells do not represent the natural extracellular material associated with each cell type and tissue. The use of “smart” scaffolds that release particular factors and/or control the temporal expression of various molecules released from the polymer could be used like dual delivery of vascular endothelial growth factor-165 and PDGF-BB, each with distinct kinetics, could produce a mature vascular network from a single, structural polymer scaffold. Another difficulty with the current materials is their lack of control over the spatial organization within the scaffold. In order to create tissues that resemble the natural structure of biological tissues, the spatial patterning of cells must be recapitulated.[71,89,90,91,92,93,94,95,96,97,98]

Stem Cell Gene Therapy

Stem cell gene therapy uses the concept that a genetically defective SC in the body could be genetically reprogrammed to become a normal SC, where the genetically corrected cell multiplies and produces more number of genetically appropriate, normal functioning cells in the body. The currently available techniques to achieve these goals are gene addition and genomic editing. For example, the most common genetic diseases of blood, such as thalassemia and sickle cell anemia could be treated either by delivery of globin transgene to HSCs or by direct repair of a specific gene mutations in HSCs.[99,100] Likewise, a genetically modified MSCs could be used to treat the genetic diseases such as osteogenesis imperfecta, Marfan's syndrome, and muscular dystrophy.[101]

Gene addition involves the delivery of corrective DNA using viral vectors (retrovirus, lentivirus) to compensate or overrides the defective gene, either by integration into one of the existing chromosomes or by incorporation of the transgene in a synthetic microchromosome.[102,103,104] Genomic editing involves DNA repair and/or homologous recombination process to correct an existing defective gene sequence in order to restore normal DNA state by delivering small DNA fragments, modified DNA polymerases and/or hybrid DNA/RNA molecules that are homologous to the defective sequence with the expectation of the bases intended DNA alteration.[105,106]

Application of Stem Cell in Dentistry

The search for MSC like cells in specific tissues resulted in the discovery of SCs in every organ and tissue including dental SCs.[107,108] For dental tissue regeneration, several potential MSC type of dental SCs have been identified in relation to the tooth and periodontal tissues. The main source of dental SCs in orofacial region includes: (i) Dental pulp SCs (DPSCs) derived from the pulp tissue of impacted tooth, (ii) SCs from human exfoliated deciduous teeth derived from the pulp tissue of a exfoliating deciduous tooth, (iii) SCs from apical papilla isolated from the soft tissue at the apices of developing permanent teeth, (iv) periodontal ligament SCs isolated from the mixed cell population of in the periodontal ligament space, (v) dental follicle precursor cells derived from an ectomesenchymal tissue surrounding the enamel organ and the dental papilla of the developing tooth germ prior to eruption.[108,109]

It has been anticipated that the SCs derived from dental tissues would be committed to dental lineage and produce only dental tissues like dentin, pulp, etc., but it has been substantiated that dental SCs have the ability to differentiate into other cell lineages. For example, DPSCs have the ability to differentiate into myogenic, adipogenic, osteogenic, chondrogenic, and neurogenic lineages. The main advantages of dental SCs over other MSCs is that ease with which teeth can be obtained and the low levels of risk and these somatic dental SCs will probably become a source of cells that will be useful not only in dentistry but also in many other regenerative therapies.[110,111,112,113]

In the therapeutic aspects, dental SCs are used in the repair of damaged dentin, pulp re-vascularization and regeneration, and for periodontal disorders.[114] In tissue engineering, whole tooth regeneration, a current dental regenerative process under progress would reduce the difficulties associated with the presently offered dental treatments such as prosthesis, implants, and tooth transplantation.[115,116,117]

Whole tooth regeneration by tissue engineering currently uses two methods; scaffold method and cell aggregates method. In scaffold method of tooth regeneration, the stem or precursor cells are arranged in proper spatial orientation using a biodegradable polymer membrane (polyglycolate/poly-L-lactate) or collagen sponge scaffolds to generate an artificial tooth germ. This tooth germ mimics tooth germ in the late stages of differentiation during normal tooth development which has proper cell polarization to achieve perfect spatial relationship in the final tooth structure (enamel, dentin, pulp, and cementum).[115,116,117] In cell aggregates method, dental epithelial tissue and mesenchymal cell pellets are dispersed in a well-controlled culture condition to create an artificial tooth germ. The tooth germ formed in this method mimics a tooth germ of the early inductive stage of tooth development where cell to cell and epithelial-mesenchymal interactions are predominant.[118,119,120,121,122,123] The hurdles associated with whole tooth regeneration with bioengineered tooth germ in the adult oral environment are fully developed neighboring tissues and lack of continuous signaling in adults in contrast to embryo where tooth germ develops along with the neighboring tissue and receives signaling molecules from there. It has been reported that a bioengineered tooth germ primordium isolated from a bioengineered tooth germ, could be transplanted into a tooth cavity after the extraction of a mandibular incisor and could develop into a tooth with typical spatial orientation of structures, such as enamel, dentin, dental pulp, root, blood vessels, periodontal ligament, and alveolar bone. These findings suggest the possibility of successful generation of whole teeth by transplantation of bioengineered tooth germs into the adult oral environment in future.[124,125]

Conclusion

Thus, we have seen the endless possibilities of what intelligent harnessing of SCs can achieve. The bioengineering technologies developed for tooth regeneration will make substantial contributions to understand the developmental process and will encourage future organ replacement by regenerative therapies in a wide variety of organs such as the liver, kidney, and heart. Further research toward the same has the potential to herald a new dawn in effective treatment of notoriously difficult diseases which could prove highly beneficial to mankind in the long run.