Stem Cells & Regenerative Medicine PDF

Stem Cells & Regenerative Medicine Dr. Farhad K.Shahian Stem Cells Characteristics They can divide and renew themselves over a long time (continuously self-renew) They have the potential to become specialized cells, such as muscle cells, blood cells, and brain cells Stem ce...

Stem Cells & Regenerative Medicine Dr. Farhad K.Shahian Stem Cells Characteristics They can divide and renew themselves over a long time (continuously self-renew) They have the potential to become specialized cells, such as muscle cells, blood cells, and brain cells Stem cells are classified according to their plasticity (developmental versatility). Totipotent Stem Cells Fertilized eggs and cells after few cell divisions These are the most versatile of the stem cell types. Have the potential to give rise to all human cells as well as entire functional organism. After four days of embryonic cell division, the cells begin to specialize into pluripotent stem cells. Pluripotent Stem Cells Like totipotent stem cells, can give rise to all tissue types. Unlike totipotent stem cells, cannot give rise to an entire organism. On the fourth day of development, the embryo forms into two layers: - an outer layer (the placenta) - an inner mass (human body) These inner cells, though they can form nearly any human tissue, cannot do so without the outer layer; so are not totipotent, but pluripotent. As these pluripotent stem cells continue to divide, they begin to specialize further. Multipotent Stem Cells Less plastic and more differentiated stem cells. They give rise to a limited range of cells within a tissue type. The offspring of the pluripotent cells become the progenitors of such cell lines as blood cells, skin cells and nerve cells. At this stage, they are multipotent. They can become one of several types of cells within a given organ. For example, multipotent blood stem cells can develop into red blood cells, white blood cells or platelets. Adult Stem Cells (Tissue-specific or somatic stem cells) An adult stem cell is a multipotent stem cell in adult humans that is used to replace cells that have died or lost function. It is an undifferentiated cell present in differentiated tissue. It renews itself and can specialize to yield all cell types present in the tissue from which it originated. Adult Stem Cells Cont’n So far, adult stem cells have been identified for many different tissue types such as hematopoetic (blood), neural, endothelial, muscle, mesenchymal, gastrointestinal, and epidermal cells Tissue-specific stem cells can be difficult to find in the human body, and they don’t seem to self-renew in culture as easily as embryonic stem cells do.  Induced pluripotent stem cells Human iPSCs were first reported in late 2007 and can generate cells characteristic of all three germ layers. iPSCs differentiate into different cell types, such as neuron, pancreas, cardiac myocytes, and renal lineage cell under appropriate condition. Reprogrammed somatic cells (pluripotency) Engineered in the lab by converting tissue-specific cells, such as skin cells, into cells that behave like embryonic stem cells. Viruses are currently used to introduce the reprogramming factors into adult cells, and this process must be carefully controlled and test. In animal studies, the virus used to introduce the stem cell factors sometimes causes cancers.  Human Tissue Sources for iPSC Dermal fibroblasts Skin keratinocytes Amniotic fluid-derived cells CD34 blood cells Mesenchymal stem cells  Mesenchymal Stem Cells (MSCs) One of the most widely studied types of adult stem cells. MSCs were first identified by Friedenstein et al. as self-renewing fibroblast-like cells in bone marrow Initially referred to as bone marrow stem cells (BMSCs) and bone marrow mesenchymal stem cells (BMMSCs). Also isolated from fat, cord blood,… MSCs can differentiate into osteogenic, chondrogenic, and adipogenic lineages.  In vitro Odontogenesis Differentiation of dental epithelial-like cells was firstly induced from the mouse ES cell using culture methods with ameloblasts serum-free medium. iPSC can differentiate into tooth related cells including dental mesenchymal cells for regeneration purpose.  Dental Mesenchymal Stem Cells Advantages: Reprogramming efficiency Multipotency Easier to obtain Lack of morbidity at the donor site iPSCs from dental cells can provide more powerful tools for regenerative application in medicine  Applications Most of the research employing dental stem cells has been directed towards regeneration of damaged tooth related structures, either partially or in its entirety Substantial number of studies in dental pulp regeneration (regenerative endodontic) Regeneration of the periodontal complex Oosseous regeneration, including craniofacial or alveolar bone Source for other tissue regeneration in medicine  Regenerative Medicine Goal: To regenerate fully functional tissues or organs that can replace lost or damaged ones due to diseases, injury and ageing Bioengineered hair follicles and the mammary gland  Regenerative Dentistry In Vitro Odontogenesis three-dimensional bioengineered teeth tooth germ generation Bioengineered teeth having: Proper tooth structure, masticatory performance, correct responsiveness to mechanical stress and neural function  Organogenesis of Tooth Tooth development relies on reciprocal tissue interactions between ectoderm-derived dental epithelium and cranial neural crest-derived mesenchyme Without epithelial cells, the mesenchymal cells differentiate into bones,… Without mesenchymal cells, the epithelial cells form skin-like cells At the initiation of tooth development, the epithelium provides the first instructional signals to the mesenchyme  Lab Induced Tooth 1) Epithelial Stem Cells (EpSC): ameloblasts 2) Mesenchymal Stem Cells (MSC): odontoblasts, cementoblasts, osteoblasts and fibroblasts of the periodontal ligament  Human Postnatal Mesenchymal Stem Cells Dental pulp stem cells (DPSCs) Stem cells from human exfoliated deciduous teeth (SHEDs) Periodontal ligament stem cells (PDLSCs) Stem cells from apical papilla (SCAPs) Dental follicle progenitor cells (DFPCs) - Each have slightly different potencies - These dental stem cells are derived from the neural crest - Bone marrow-derived MSCs are derived from the mesoderm  Dental Pulp Stem Cells (DPSCs) DPSCs are the first tooth derived stem cells and are mesenchymal type of cells inside dental pulp Differentiate into: osteoblast, smooth muscle cells, adipocyte-like cells, neuron, dentin, and dentin-pulp-like complex They were also shown to have chondrogenic potentials in vitro. Overall, DPSCs are more suitable than BMSCs for mineralized tissue regeneration Considering the origin of dental pulp tissue, it is not surprising that DPSCs are capable of differentiating into both neural and vascular endothelial cells  Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) SHEDs are progenitor cells isolated from the pulp remnant of exfoliated deciduous teeth. More proliferation rate and higher capability for differentiation than BMSCs and even DPSCs in several studies Osteoblast, odontoblast, adipocyte, and neural cells have been reported to be differentiated from SHEDs Periodontal Ligament Stem Cells (PDLSCs) PDL has neural crest cell origin However, PDLSCs exhibit stem cell characteristics similar to MSCs PDLSCs can differentiate into osteoblast, cementoblasts, adipocytes, chondrocyte, periodontal ligament and cementum-like tissue in vivo  Stem Cells from Apical Papilla (SCAPs) SCAPs are cells isolated from the root apex of developing tooth They present the characteristics of MSCs and can differentiate into osteoblast, adipocyte, chondrocyte, and neuron under appropriate conditions  Dental Follicle Progenitor Cells (DFPCs) DFPCs are stem cells extracted from dental follicle surrounding tooth germ in early tooth formation stages The dental follicle is an ectomesenchymal cell condensation and harbors heterogeneous population of cells comprising periodontium. They are also known to be differentiated into osteoblast, adipocyte, chondrocyte, and neuronal cells  Dental Epithelial Stem Cells They are not readily found in postnatal dental tissues Ameloblasts are lost upon tooth eruption Recent studies show that at the stage of crown formation, epithelial cell rest of malassez (ERM) can differentiate into ameloblast-like ERM combined with dental pulp cells expressed cytokeratin and amelogenin proteins in vitro. Eight weeks after the transplantation: - Enamel-like tissues showed positive staining for amelogenin, indicating the presence of well-developed ameloblasts in the implants Odontogenic potential Capability of a tissue to induce gene expression in an adjoining tissue and to commence tooth genesis Odontogenic potential is present initially in the dental epithelium and in the later stages it shifts to the mesenchyme (Mice studies) Odontogenic competence Capability of a tissue to reciprocate to odontogenic signals and to support tooth genesis Only the mesenchyme derived from the cranial neural crest is capable of odontogenic competence  Recombining early dental epithelium with non-neural crest derived mesenchyme = NO TEETH Recombining the epithelium derived from the first branchial arch and mesenchyme of second branchial arch (neural crest derived cells) or with premigratory trunk neural crest = tooth formation The dental mesenchyme from mouse embryonic cap and bell stage teeth can instruct tooth development when combined with non-dental epithelium Is Human Teeth Regeneration a Prospective Clinical Reality or a Fantasy?  Biological Tooth Repair and Regeneration Numerous animals (most nonmammalian species) are endowed with the ability to continually replace lost teeth throughout life via de novo formation of tooth germs (polyphyodonty) In various animal species (e.g., mice, voles), it is possible to replace physically worn teeth parts of varying teeth types (mouse incisors, vole molars) with stem cells  Partial tooth repair Gradual enamel and dentin loss and pulp exposure induces living odontoblasts to restart the production of reactionary dentin. Large lesion: - Death of odontoblasts - TGFβ growth factors are liberated from the destructed extracellular matrix - These growth factors recruit pulp perivascular stem cells, which migrate to the injured region and produce reparative dentin. Identification of various enamel proteins (mainly amelogenin) in odontoblasts could lead to the ability to synthesize and deposit dentin and enamel during tooth repair  Various enamel proteins (mainly amelogenin) are discovered in odontoblasts (ability to synthesize and deposit dentin and enamel) Adult stem cells and their cultivation/multiplication in a biodegradable scaffold of shape complementary to the lost tooth part. Conditions should be chosen to guide odontoblast and ameloblast differentiation and the orderly production of materials needed to correctly replace the missing ones. Thus far, there have been numerous in vitro and in vivo studies performed on partial tooth repair that have generated exciting and strongly promising results. Central giant cell tumors (CGCTs) are uncommon, benign and are locally destructive osteolytic lesions. Pediatric patients 5 to 15 years of age. Multiple noninvasive modalities of treatment (intra-lesional steroids, interferon, calcitonin, and denosumab). Enucleation and curettage or resection is a curative surgery.  This case report describes a pediatric patient who was diagnosed with an aggressive CGCT of the left mandible encompassing the right angle to the condyle. The lesion became refractory to noninvasive treatments and immediate resection and reconstruction was performed using principles of tissue engineering. 5 year follow up, the patient showed normal morphology and growth of his mandible, but surprisingly developed a left mandibular third molar (tooth 17) in the site of the mandibular resection and reconstruction. Show the spontaneous development of teeth in a human reconstructed mandible, contributing evidence toward the functional matrix theory of mandibular growth and ectodermal origin of teeth.  Replacement of a whole tooth To successfully achieve this goal, the scientist needs: Tooth-related cells from each patient, preferably isolated stem cells from the corresponding teeth tissues that are endowed with odontogenic potential. Odontogenic epithelial cells of human origin: - Impacted or developing third molar, dental lamina in postnatal life, ERM (epithelial cell rests of Malassez), and in reduced enamel epithelium. The experimental recombination of either dissociated epithelial and mesenchymal cells or intact dental epithelium and mesenchyme creates tooth-like structures that have an organized apposition of the main tooth constituents. Culture techniques Permit a fast expansion that would yield the needed cell quantities to condition the microenvironment and allow for cell-cell interactions that would lead to purposeful cell differentiation. Adequate microenvironment Supporting 3D tooth-like growth, leading to the production of a genuine tooth-like bud or even a tooth replica. Utilization of an artificial, tooth-shaped biodegradable scaffold. Various scaffold materials have already been employed for this purpose and have yielded promising results. Surgical technique: The ability to implant the bioengineered bio-tooth germ into the prepared empty alveolar socket under conditions that permit the development of the root, periodontal ligament and osteointegration. This goal has already been achieved, proving that the jaw of an adult animal can accept a bioengineered tooth bud, nurture it and cooperate with it in a way that allows for tooth growth, morphogenesis and eruption. Novel three-dimensional cell manipulation methods for whole tooth regeneration Engraftment of Bioengineered Tooth A) Dissociated mesenchymal cells at a high density are injected into the center of a collagen drop. Dissociated tooth germ-derived epithelial cells are subsequently injected into the drop adjacent to the mesenchymal cell aggregate Within 1 day of organ culture, cell-to-cell compaction was observed B) By transplanting a bioengineered tooth germ into a subrenal capsule for 30 days Bioengineered tooth comprising a mature tooth with the correct structural components such as enamel (E), dentin (D), periodontal ligament (PDL) and alveolar bone (AB) Transplantation of Bioengineered Tooth Germ Transplantation of Bioengineered Tooth Germ A) Transplanted bioengineered tooth germ erupted and reached the occlusal plane with the opposing lower first molar (49 days after transplantation) B) GFP-labelled bioengineered tooth erupted in the oral environment of adult mice C) A bioengineered tooth unit was engrafted by bone integration and reached the occlusal plane with the opposing upper first molar at 40 days post transplantation. Bioengineered tooth germ implants Develop into the correct tooth structure in an oral cavity Successfully erupt 37 days after transplantation (Mice) Reaches the occlusal plane and achieves occlusion with the opposing tooth from 49 days onwards Bioengineered mature tooth transplant Occludes with the opposing upper first molar after 40 days Maintains the periodontal ligament originating from the bioengineered tooth unit through successful bone integration Both system: The enamel and dentin hardness of the bioengineered teeth components were in the normal range