![A] Stem Cells
Defined as having three important properties:
Ability to self-renew,
Ability to differentiate into a number of different cell types,
Ability to easily form clonal populations
Autologous, Allogenic](https://image.slidesharecdn.com/1principlesoftissueengineering-200716071253/85/1-principles-of-tissue-engineering-7-320.jpg)
![B] Native Targeted Progenitor Cells
Tissue specific unipotent cells derived from most organ
Already programmed to become the cell type needed, without
any extra-lineage differentiation
obtained from the specific organ to be regenerated, expanded,
and used in the same patient without rejection](https://image.slidesharecdn.com/1principlesoftissueengineering-200716071253/85/1-principles-of-tissue-engineering-18-320.jpg)
![C] Somatic Cell Nuclear Transfer
Removal of an oocyte nucleus in culture, followed by its
replacement with a nucleus derived from a somatic cell obtained
from a patient
Two types of cloning
Reproductive cloning
Therapeutic cloning
Both involve the insertion of donor DNA into an enucleated
oocyte to generate an embryo that has identical genetic material
to its DNA source.](https://image.slidesharecdn.com/1principlesoftissueengineering-200716071253/85/1-principles-of-tissue-engineering-22-320.jpg)
![D] Reprogramming
(Induced Pluripotent Stem Cells)
Involves de-differentiation of adult somatic cells to produce
patient-specific pluripotent stem cells, eliminating the need to
create embryos
genetically identical to the somatic cells and would not be
rejected
iPS cells possessed the characteristics of
self-renewing embryonic stem cells
expressed genes specific for embryonic stem cells
generated embryoid bodies in vitro
teratomas in vivo](https://image.slidesharecdn.com/1principlesoftissueengineering-200716071253/85/1-principles-of-tissue-engineering-27-320.jpg)
#1 principles of tissue engineering
- 1. Principles of Tissue Engineering Dr Roshan V Shetty Senior Resident In Urology AJ IMS & RC
- 2. Why? Organ transplantation remains a mainstay of treatment for patients with severely compromised organ function. Despite initiatives to increase the availability of transplant organs, however, the number of patients in need of treatment far exceeds the organ supply, and this shortfall is expected to worsen as the global population ages. One alternative treatment approach is regenerative medicine.
- 3. What? “An interdisciplinary field of research and clinical applications focused on repair, replacement or regeneration of cells, tissue or organs to restore impaired function from any cause(ie. Congenital defects, disease, trauma, ageing)” Combines converging technological approaches, both existing and newly emerging, moving it beyond traditional transplantation and replacement therapies. Approaches often aim to stimulate and support the body’s own self healing capacity
- 4. How? Approaches may include: Use of soluble molecules Gene therapy Stem and progenitor cell therapy Tissue engineering Reprogramming of cell and tissue types
- 5. Regenerative Medicine: Strategies for Tissue and Organ Reconstitution Cell-based therapies Biomaterial based therapies The use of biomaterials (scaffolds) alone, wherein the body’s natural ability to regenerate is used to orient or direct new tissue growth) Combined (tissue engineering) The use of scaffolds seeded with cells to create tissue substitutes.
- 6. Sources of Cells for Therapy A. Stem Cells B. Native Targeted Progenitor Cells C. Therapeutic Cloning (Somatic Cell Nuclear Transfer) D. Reprogramming (Induced Pluripotent Stem Cells)
- 7. A] Stem Cells Defined as having three important properties: Ability to self-renew, Ability to differentiate into a number of different cell types, Ability to easily form clonal populations Autologous, Allogenic
- 8. Stem Cells Three broad categories Embryonic stem cells Fetal and neonatal amniotic fluid and placenta Adult stem cells
- 9. 1)Embryonic Stem Cells Human embryonic stem cell- 1981,Martin Pluripotent cells- skin to neurons form embryoid bodies form teratomas in vivo Found in the inner cell mass of the human embryo Able to differentiate into all cells of the human body, excluding placental cells ( Only cells from the morula are totipotent ) maintain undifferentiated state for at least 80 passages
- 10. Embryonic Stem Cells Ideal resource for regenerative medicine Ability to self-renew indefinitely Ability to differentiate into cells from all three embryonic germ layers Limited clinical application : Allogenic Ethical issues : results in destruction of embryos
- 11. Embryonic Stem Cells Alternate techniques without destroying embryos single-cell embryo biopsy Arrested embryos Altered nuclear transfer
- 12. 2)Perinatal Stem Cells Umbilical cord blood collected at the time of birth more immature than adult bone marrow stem cells autologous and allogeneic cell therapy mostly used for hematopoietic applications
- 13. Perinatal Stem Cells Wharton jelly surrounds the cord mesenchymal in origin limited multipotentiality not been used clinically
- 14. Perinatal Stem Cells Amniotic fluid and placenta Capable of extensive self-renewal Give rise to cells from all three germ layers Properties somewhere between those of embryonic and adult stem cell Do not form teratomas For self-use, avoid the problems of rejection Obtained either from Amniocentesis or Chorionic villous sampling in the developing fetus or From the placenta at the time of birth.
- 15. 3)Adult Stem Cells Many types of adult stem cells have been identified in organs throughout the body and are thought to serve as the primary repair entities for their corresponding organs Limited clinical application : Some cells have very low proliferative capacity in vitro Functionality is reduced after the cells are cultivated Isolation of cells has also been problematic
- 16. Adult Stem Cells Mesenchymal stem cell Multipotent adult progenitor cell Derived from bone marrow stroma Can differentiate in vitro into numerous tissue types
- 17. Adult Stem Cells Hematopoietic stem cells Best understood Used for decades for hematopoietic disorders Adipose-derived stem cells Could give rise to multiple lineages Differentiation into urologic cells Used experimentally to improve bladder function Urine-derived stem cells have also been proposed for genitourinary reconstruction
- 18. B] Native Targeted Progenitor Cells Tissue specific unipotent cells derived from most organ Already programmed to become the cell type needed, without any extra-lineage differentiation obtained from the specific organ to be regenerated, expanded, and used in the same patient without rejection
- 19. Native Targeted Progenitor Cells Exploring the conditions that promote differentiation and/or self- renewal, it has been possible to overcome some of the obstacles that limit cell expansion in vitro donor tissue is dissociated into individual cells which are either implanted directly into the host or expanded in culture, attached to a support matrix, and re-implanted after expansion.
- 20. Native Targeted Progenitor Cells System of urothelial cell harvesting was developed that does not use any enzymes or serum. Has a large expansion potential Possible to expand a urothelial strain from a single specimen (1cm2)to one covering a surface area of one football field(4202m2) within 8 weeks.
- 21. Native Targeted Progenitor Cells Bladder, ureter, and renal pelvis cells can equally be harvested, cultured, and expanded Not all human cells can be grown or expanded in vitro e.g. Liver, nerve, and pancreas
- 22. C] Somatic Cell Nuclear Transfer Removal of an oocyte nucleus in culture, followed by its replacement with a nucleus derived from a somatic cell obtained from a patient Two types of cloning Reproductive cloning Therapeutic cloning Both involve the insertion of donor DNA into an enucleated oocyte to generate an embryo that has identical genetic material to its DNA source.
- 23. Somatic Cell Nuclear Transfer Reproductive cloning Reproductive cloning: the embryo is implanted into the uterus of a pseudo-pregnant female to produce an infant that is a clone of the donor Eg : birth of a sheep named Dolly in 1997
- 24. Somatic Cell Nuclear Transfer Therapeutic cloning Embryo is used to generate blastocysts that are explanted and grown in culture rather than in utero Embryonic stem cell lines can then be derived from blastocysts, which are allowed to grow only up to a 100-cell stage At this time the inner cell mass is isolated and cultured Resulting in embryonic stem cells that are genetically identical to the patient
- 25. Somatic Cell Nuclear Transfer Therapeutic cloning Pluripotent nuclear transferred embryonic stem cells have been derived from fibroblasts, lymphocytes, olfactory neurons perfectly matched to the patient’s immune system and no immunosuppressants would be required However, mitochondrial DNA contained in the oocyte could lead to immunogenicity after transplantation
- 26. Somatic Cell Nuclear Transfer Therapeutic cloning Ethical considerations regarding the potential of the resulting embryos to develop into cloned embryos Limitations that require further improvement not been shown to work in humans to date
- 27. D] Reprogramming (Induced Pluripotent Stem Cells) Involves de-differentiation of adult somatic cells to produce patient-specific pluripotent stem cells, eliminating the need to create embryos genetically identical to the somatic cells and would not be rejected iPS cells possessed the characteristics of self-renewing embryonic stem cells expressed genes specific for embryonic stem cells generated embryoid bodies in vitro teratomas in vivo
- 28. Reprogramming (Induced Pluripotent Stem Cells) Generated human iPS cells are similar to hESCs in terms of morphology, proliferation, gene expression, surface markers, and teratoma formation Cells have shown great promise in the understanding of human disease, as well as the use of these cells for therapy. Like embryonic stem cells, the iPS cells also form teratomas, and this has limited their therapeutic potential.
- 29. Summary Stem Cells Reproductive/Therapeutic Cloning (SCNT) Reprogramming (iPSC) Native Targeted Progenitor Cells Embryonic stem cells Perinatal Stem cells Adult stem cells
- 30. Biomaterials and Vascularization for Genitourinary Regenerative Medicine Biomaterials function as an artificial ECM and elicit biologic and mechanical functions of native ECM Biomaterials facilitate : localization and delivery of cells and/or bioactive factors define a 3D space for formation of new tissues with appropriate structure guide development of new tissues with appropriate function
- 31. Design and Selection of Biomaterials Must be capable of controlling the structure and function of the engineered tissue in a predesigned manner by interacting with transplanted cells and/or host cells Ideal biomaterial : biocompatible promote cellular interaction and tissue development possess proper mechanical and physical properties
- 32. Design and Selection of Biomaterials Biocompatible should be biodegradable and bioresorbable to support the reconstruction of a completely normal tissue without inflammation degradation rate and the concentration of degradation products in the tissues surrounding the implant must be at a tolerable level
- 33. Design and Selection of Biomaterials Promote cellular interaction and tissue development should provide an appropriate regulation of cell behavior to promote the development of functional new tissue This is regulated by multiple interactions with the microenvironment, including interactions with cell adhesion ligands and with soluble growth factors
- 34. Design and Selection of Biomaterials Promote cellular interaction and tissue development provide temporary mechanical support sufficient to withstand in vivo forces and maintain a potential space for tissue development The mechanical support should be maintained until the engineered tissue has sufficient mechanical integrity to support itself
- 35. Design and Selection of Biomaterials Possess proper mechanical and physical properties large ratio of surface area to volume is often desirable to allow the delivery of a high density of cells A high-porosity, interconnected pore structure with specific pore sizes promotes tissue ingrowth from the surrounding host tissue
- 36. Types of Biomaterials Three classes of biomaterials have been used Naturally derived materials, such as collagen Acellular tissue matrices, such as bladder and small-intestinal submucosa Synthetic polymers, such as polyglycolic acid (pga), polylactic acid (pla), and poly(lactic-co-glycolic acid) (plga).
- 37. Types of Biomaterials 1) Collagen Collagen is the most abundant and ubiquitous structural protein in the body, and it may be Readily purified with an enzyme treatment Intermolecular cross-linking reduces the degradation by making it less susceptible to an enzymatic attack Contains cell-adhesion domain sequences that exhibit specific cellular interactions
- 38. Types of Biomaterials Can be processed into a wide variety of structures such as sponges, fibers, and films
- 39. Types of Biomaterials 2)Alginate Polysaccharide isolated from seaweed Used as an injectable cell delivery vehicle and a cell immobilization matrix Copolymers of d-mannuronate and l-guluronate Physical and mechanical properties are in proportion to the length of poly-guluronate block
- 40. 3)Bioprinting Natural materials such as have been used as “bio-inks” in bioprinting technique based on inkjet technology Materials can be “printed” into a desired scaffold shape Living cells can also be printed using this technology A three-dimensional construct containing a precise arrangement of cells, growth factors, and ecm material can be printed
- 41. Types of Biomaterials 4)Acellular tissue matrices collagen-rich matrices prepared by removing cellular components from tissues Slowly degrade after implantation and are replaced and remodeled by ECM proteins synthesized and secreted by transplanted or ingrowing cells Mechanical properties of the acellular matrices are not significantly different from those of native bladder submucosa
- 42. Types of Biomaterials 5)Synthetic polymers Polyesters of α-hydroxy acids : PGA, PLA, and PLGA Degradation products are nontoxic, natural metabolites that are eventually eliminated from the body in the form of CO2 and water drawback of the synthetic polymers is lack of biologic recognition. Needs incorporation of cell recognition domains
- 43. Types of Biomaterials Thermoplastics: they can easily be formed into a three- dimensional scaffold with a desired microstructure, gross shape, and dimension by various techniques
- 44. Vascularization A limiting factor for the engineering of tissues is that cells cannot be implanted in volumes exceeding 3 mm3 Vascularization of the regenerating cells is essential to provide nutrition and gas exchange beyond this maximal diffusion distance
- 45. Vascularization Two different processes: Vasculogenesis, the in situ assembly of capillaries from undifferentiated endothelial cells (ECs), and Angiogenesis, the sprouting of capillaries from preexisting blood vessels
- 46. Vascularization Vasculogenesis ECs are generated from precursor cells, called angioblasts, ECs form the vessel primordia and aggregates, have no lumen; a nascent endothelial tube is formed, composed of polarized ECs; a primary vascular network is formed from an array of nascent endothelial tubes; and pericytes and vascular smooth muscle cells are recruited
- 47. Vascularization Angiogenesis vasodilatation of the parental vessel, reducing the contact between adjacent ECs; degradation of the basement membrane EC migration and proliferation to form a leading edge of the new capillary; generation of the capillary lumen and formation of a tube like structure; basement membrane synthesis; and recruitment of pericytes and vascular smooth muscle cells
- 48. Vascularization Three approaches incorporation of angiogenic factors before implantation, to attract host capillaries and to enhance neovascularization seeding ECs with other cell types in before implantation eg. smooth muscle cells and Ecs in penile corporeal tissue Pre-vascularization of the matrix before cell seeding to form vascular network, providing sufficient tissue perfusion
- 49. Stem cells Biomaterials Vascularisation Engineered Tissue for Regeneration
- 50. Thank You !