Stem cells are commonly classified based on the developmental stage from which they are isolated, although this has been a source of debate amongst stem cell scientists. A common approach classifies stem cells into three different groupings: Embryonic Stem Cells (ESCs), Umbilical Cord Stem Cells (UCBSCs) and Adult Stem Cells (ASCs), which include stem cells from bone marrow (BM), fat tissue (FT), engineered induced pluripotent (IP) and peripheral blood (PB). By definition stem cells are progenitor cells capable of self-renewal and differentiation hypothetically "ab infinitum" into more specialized cells and tissue. The main intent of this study was to determine and characterize the different sub-groups of stem cells present within the human PB-SCs that may represent a valid opportunity in the field of clinical regenerative medicine. Stem cells in the isolated mononucleated cells were characterized using a multidisciplinary approach that was based on morphology, the expression of stem cell markers by flowcytometry and fluorescence analysis, RT-PCR and the capacity to self-renew or proliferate and differentiate into specialized cells. This approach was used to identify the expression of hematopoietic, mesenchymal, embryonic and neural stem cell markers. Both isolated adherent and suspension mononucleated cells were able to maintain their stem cell properties during in-vitro culture by holding their capacity for proliferation and differentiation into osteoblast cells, respectively, when exposed to the appropriate induction medium.

In this paper we describe an approach that aims to provide fundamental information towards a scientific, biomechanical basis for the use of natural coral scaffolds to initiate mesenchymal stem cells into osteogenic differentiation for transplant purposes. Biomaterial, such as corals, is an osteoconductive material that can be used to home human derived stem cells for clinical regenerative purposes. In bone transplantation, the use of biomaterials may be a solution to bypass two main critical obstacles, the shortage of donor sites for autografts and the risk of rejection with allograft procedures. Bone regeneration is often needed for multiple clinical purposes for instance, in aesthetic reconstruction and regenerative procedures. Coral graft Porites lutea has been used by our team for a decade in clinical applications on over a thousand patients with different bone pathologies including spinal stenosis and mandibular reconstruction. It is well accepted that human bone marrow (hBM) is an exceptional source of mesenchymal stem cells (MSCs), which may differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. Isolated MSCs from human bone marrow were induced into osteoblasts using an osteogenic medium enriched with two specific growth factors, FGF9 and vitamin D2. Part of the cultured MSCs were directly transferred and seeded onto coral scaffolds (Porites Lutea) and induced to differentiate into osteoblasts and part were cultured in flasks for osteocell culture. The data support the concept that hBM is a reliable source of MSCs which may be easily differentiated into osteoblasts and seeded into coral as an optimal device for clinical application. Within this project we have also discussed the biological nature of MSCs, their potential application for clinical transplantation and the prospect of their use in gene therapy.

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs' in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7 days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.

It is well accepted that human umbilical cord blood (UCB) is a source of mesenchymal stem cells (MSCs) which are able to differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. The aim of this study was to isolate MSCs from human UCB to determine their osteogenic potential by using different kinds of osteogenic medium. Eventually, only those MSCs cultured in osteogenic media enriched with vitamin D(2) and FGF9, were positive for osteocalcin by RT-PCR. All these cells were positive for alizarin red, alkaline phosphatase and Von Kossa. The results obtained from RT-PCR have confirmed that osteogenesis is complete by expression of the osteocalcin marker. In conclusion, vitamin D(2), at least in vitro, may replace vitamin D(3) as an osteogenic stimulator factor for MSC differentiation.

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