Stem cells are the natural reservoir for growth, tissue turnover and repair they are “undifferentiated cells which can undergo asymmetric division, proliferation and generate cells which are differentiating to adult progeny” [1] Principal characteristics are the ability of self – renewal (by this of replenishing the stem cell pool) the capability of proliferation (up to 50 passages in culture without undergoing chromosomal abnormality and without showing signs of senescence) and potential to differentiate into various lineages. They are classified as pluripotent (embryonic stem cells) or multipotent (postnatal somatic stem cells or adult stem cells ASC) Pluripotent embryonic stem cells are capable of differentiating into any type of tissue, while adult stem cells are, in natural conditions, restricted to the tissue they reside. The pluripotent ESC is derived from the inner cell mass of the blastocyst and has the ability to give rise to all three embryonic germ layers; ectoderm, endoderm, and mesoderm. ASCs are involved in tissue homeostasis, tissue regeneration and cell replacement owing to injury or natural death.
The origin of adult stem cells in mature tissues is still under discussion. The question of whether there are universal stem cells or stem cells resting in the individual tissue has not yet been answered. There might be adult stem cells circulating in the blood stream or located in the blood vessels and able to populate different tissues. Several researchers have noted that dividing
Cells in adult tissue often appear near a blood vessel, such as candidate stem cells in the hippocampus and pericytes in the blood vessels [2] another hypothesis is that the stem cells
reside in the tissue from the embryonic development. In some tissues it is clear that these stem cells are located in a special microenvironment known as the “niche”. The location and nature of this niche can vary depending on the type of tissue. The niche is assumed to be a dynamic structure keeping the stem cells in quiescence and contributing to the activation of stem cells when required. Stem cells, the natural reservoir for growth, tissue turnover and repair they are “undifferentiated cells which can undergo asymmetric division, proliferation and generate cells which are differentiating to adult progeny” [3] Principal characteristics are the ability of self – renewal (by this replenishing the stem cell pool [4] the capability of proliferation (up to 50 passages in culture without undergoing chromosomal abnormality and without showing signs of senescence) and potential to differentiate into various lineages. They are classified as pluripotent (embryonic stem cells) or multipotent (postnatal somatic stem cells. Pluripotent embryonic stem cells are capable of differentiating into any type of tissue, while adult stem cells are, in natural conditions, restricted to the tissue they reside. [1].
Mesenchymal stem cells isolated from bone marrow or from other tissues via their propensity to adhering to the plastic walls of culture containers, can provide a rich source of cells for cartilage repair procedures. MSCs are a heterogeneous population of multipotent stem cells that are frequently described as fibroblast-like forming colonies, with the ability to differentiate into multi-mesenchymal lineage (such as osteoblasts, chondrocytes and adypocites) and myoblast under defined culture conditions. MSCs isolated from bone marrow by needle aspiration have been considered, until recently, the easiest accessible and richest source of precursor cells. However, resident MSCs from adipose tissue, muscle, synovium, umbilical cord blood, amniotic fluid 5], and trabecular bone [6] are in study for efficiency and promise to generate less invasive harvesting procedures. Human MSCs can be culture expanded more than 1 billion fold with preserving their multilineage potential. Stem cells have the advantage to be of unlimited supply, their high proliferative rate allows for expansion in culture to large numbers, making them attractive as cells source for cartilage regeneration. Moreover, it has been postulated that induction of stem cells to various mesoderm lineages will produce a tissue more closely resembling the desired one by recapitulating embryonic development. Another postulated advantage of using stem cells for tissue engineering strategies is that they could be used in allergenic transplantation. When cultured with allogeneic lymphocytes, human MSC do not induce lymphocyte reaction are able to suppress an ongoing mixed lymphocyte reaction.
Besides being a valuable source for cell-based therapies, stem cells may be also used as carrier for delivery of bioactive molecules through genetic manipulation. MCSs transduced by recombinant adenoviral, retroviral, lentiviral and foamyviral vectors are in study; marrow-derived MSCs genetically modified to express TGF-ß1 or BMP have been found to undergo chondrogenesis in aggregate culture. Some first studies have been performed applying periosteal, perichondral or marrow-derived MSCs mediated gene delivery for cartilage repair in vivo.
There are three different approaches to regeneration of functional tissue in current engineering procedures, using postnatal stem cells, as follows [6]: expansion of a cellular population ex vivo before implantation into the host; ex vivo generation of a tissue or even organ for transplantation; devices or substances for in vivo activation of local or distant stem cell in order to induce tissue repair. The first fundamental tissue engineering approach implies delivery and integration of functional cells capable to sintethyse cartilage matrix specific proteins in order to restore pathologically altered structure and function. ACI represents technically the first cell therapy for cartilage repair, using adult differentiated cells. Use of progenitor cells as a source for cartilage regeneration intents to avoid the presented inconvenient. Most of the research on stem cells has focused on either embryonic or postnatal stem cells. Embryonic stem cells are isolated from early embryo and are considered to be pluripotent, capable to differentiate to various lineages.
Embryonic stem cells (ESCs) are derived from inner cell mass of early blastocyst, (morulae) from the pre inplantation embryo. Under specific culture condition they can be induced to differentiate into cells of all three germ layers (ectoderm, mesoderm and endoderm). ESCs can also be grown as undifferentiated cells, providing a valuable rich supply of uncommitted cells. In vitro chondrocyte differentiation of ESCs has been reported [1] the optimal culture conditions for obtaining pure populations are still under debate. There are several issues to be addressed when using embryonic stem cells for tissue engineering: eventuality of human embryo manipulation is one of the most debated ethical issues of the last decade. ESC are obtained from embryos derived from unsuccessful IVF (in vitro fertilization) which would otherwise be discarded, however the methods is prone to offer a background for human tissue commercialization and therefore banned in several countries. Another issue is the eventuality of using cells from an allogeneic source.
Cells to differentiate from embryos can display unknown immunogenic potential raising the threat of GVHD (graft versus host disease) similar to other foreign tissue transplantation and therefore requiring immune suppression, one of the disadvantages of organ transplantation RM is committed to avoid. On the other hand, ESC are the most versatile source considered for RM because of their multipotentiality, therefore regarded as having unlimited capacity for cell and tissue replacement therapy. Besides ethical problems raised by their manipulation, obviously ESC must be used in form of allografts [7].
Attempts are ongoing to therapeutically cloning human embryos in order to generate autologus multipotent cells, however; the major social and ethical concerns they raised and the uncertainty about their behaviour after implantation, makes the attempt to using embryonic stems as a source for future regenerative therapies, a remote perspective. Moreover, another important draw back in using ESC for everyday clinical therapies is the tendency of implanted embryonic stem cells to forming teratomas. Tumours of high degree of malignancy and invasiveness, teratomas form both in the culture dish of after ESC implantation in the animal model. Malignant transformation of a grafted tissue for a non-life threatening disease represents an unacceptable complication. Given the exposed issues, the interest focused on the use of postnatal stem cells.
6.2.4. Postnatal adult stem cells Current data indicate the existence of two types of postnatal stem cells. Tissue non specific stem cells are haematopoietic in origin and can differentiate into different blood lineages. Tissue-specific stem cells differentiate into cells of residing tissue (even though a rather limited ability to turn to other lineages is possible) First described and first to be used were mesenchymal stem cells from bone marrow progenitor cells, BMMSCs BMMSCs reside in hematopoietic active bone marrow as supportive cells contributing to functional modulation of hematopoietic niche. However, accumulated evidence exists about the presence of populations of undifferentiated multipotent cells involved in tissue regeneration after trauma, disease or even ageing in various structures like adipose tissue , skeletal muscle, umbilical cord blood, synovium, periosteum, tendon, menisci [6].
Mesenchymal stem cells MSCs are multipotent stem cells with the ability to differentiate in
to a variety of mesenchymal lineage cells such as chondrocytes, osteoblasts, adypocites, tenoblasts or myoblasts Their high proliferative capacity allow ex vivo expansion without loss of phenotype and potentiality therefore being considered as cell sources for regenerative strategies. MSCs are characterized by their ability to adhere to the plastic of culture dish and to forming colonies (colony forming unit-fibroblast, CFU-F). Submitted to distinct and specific culture condition and differentiation media, MSCs are capable of switch towards multiple lineages. They are considered to be positive for a set of surface markers: CD44, CD71, CD90, CD106, CD120a CD124 and negative for known hematopoietic lineage markers as CD44, CD34, and CD45 [8]. Another important characteristic is the presence of a distinct pattern of secreted cytokines like LIF (Leukemia Inhibitory Factor) IL-6, IL-1, granulocyte-macrophage colony stimulating factor (GM-CSF)). In bone marrow the frequency of MSCs in nucleated cells varies between 0, 0001-0, 01% and decreases with the age. The ideal source remains controversial, reports exists about higher chondrogenic potential of synovial tissue [9] than adipose tissue or low chondrogenic capacity of skeletal muscle. However, regardless the source, the chondrogenetic potential of MSCs has been demonstrated and induction of in vitro chondrogenesis follows a standard procedure. Chondrogenesis is induced in high-density culture in vitro in serum free media and in the presence of transforming growth factor ß (TGF-ß1 or TGF-ß3), in hypoxic conditions. After a variable period of time (14.21 days) the presence within the culture system of chondrocyte-like phenotype of progeny can be detected. Chondrocytes derived from MSCs are characterized by expression of cartilage-specific genes encoding ECM cartilage specific protein: collagen type II (Col1A) collagen IX, aggrecan, or master key regulators of chondrogenesis (sox-9). In MSCs –derived chondrocytes, the gene-expression levels for cartilage molecules was found to be below the level of articular cartilage chondrocytes and closer to intervertebral disc derived adult cells. MSC populations with comparable proliferative and differentiation potential with BMMSCs, have been isolated from different mensenchymatous tissues and reported as being able of generating the amount and quality of cells required for clinical therapies.
Adipose layer appear to be the richest source of progenitor cells, adipose derived MCSs (ADSCs) are relatively easy to obtain with site pathology, are more abundant within the tissue, therefore regarded as an important cell source for regenerative strategies already used in several going on clinical trials.[9] Adipose derived stem cells are regarded as a convenient source of cells because their convenient availability. They can be collected during cosmetic procedures (mechanical liposuction, surgical or ultrasound adipose tissue ablation, during bariatric surgery) with minor surgery or adjacent to other surgical gestures and can provide an important number of autologus cell to be stored for future regenerative procedures.
Stem cells derived from articular synovial layer [10] and from trabecular bone [11] have been reported as displaying important biological characteristics attractive for cartilage regeneration purposes. The available data about the presence of MSCs within different joint components and periarticular tissues, existence of progenitors within the cartilaginous tissue and evidence of the presence of progenitor cells capable of migration and chondrogenetic differentiation within the osteoarthritic cartilage induced the reasoning that OA cartilage could derive progenitors to be expanded in vitro and further use for regenerative therapies.
Exposed to differentiation specific high density culture systems (pellet culture or alginate beads) MSCs of various origins undergo commitment to chondrogenic lineage. Cells acquire a rounded body and group in a delimited matrix as chondrons in adult hyaline cartilage. However, the amount of forming matrix proteins and their mechanical properties are lower than that produced by chondrocytes in similar conditions. Applying mechanical stress during culturing MSCs in vitro results in increased matrix formation leading to conclusion that mechanical loading could be considered as a valuable tool to improve stable cartilage formation.
Mechanical preconditioning of stem cell derived chondrocytes is considered a prerequisite for obtaining cells or structures with enhanced functional adaptability while in vivo.
Induced pluripotent stem cells. Reversing biology by means of advanced science has always been a human dream. Emerging field of regenerative medicine seems to have brought this dream one step closer.
Few areas of biology currently garner more attention than the study of human pluripotent stem cells (hPSC). This interest has arisen because of their potential to form the basis of cellular therapies for diseases affecting organ systems with limited regenerative capacity, to provide enhanced systems for drug screening and toxicity testing as well as to gain insight into early human development obviating the need for human embryos. There are currently two major methods for generating cells with pluripotent properties. The first involves isolating the inner cell mass from an early human blastocyst and culturing the resulting cells in appropriate culture conditions (see below) to generate human embryonic stem cells (hESC). The second involves artificially expressing a defined number of factors in somatic cell types, which, with the appropriate culture conditions, causes the cells to be reprogrammed into induced pluripotent stem cells (iPSC)Pioneering work has revealed that terminally differentiated somatic cells can be reprogrammed to generate induced pluripotent stem (iPS) cells via overexpression of a defined set of transcription factors. These iPS cells are morphologically and phenotypically similar to embryonic stem (ES) cells and thus offer exciting possibilities in stem cell research and regenerative medicine. Moreover, iPS cells are useful tools for studying the pathogenesis of human disease, for drug discovery and toxicity screening [12].
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