Advances in regenerative medicine have been concentrated in Stem cells research and its clinical applications. Embryonic and adult stem cells have been widely studied and characterized; cells lines and therapies have been developed since the first evidence of the existence of stem cells was obtained in 1963. This review examines the history and evolution of the stem cells research and gives understanding concepts on the topic.
Over the past years, advances in stem cells therapy have occurred as a source of regeneration and repairing of damaged tissue. Stem cells characteristics of unlimited self-renewal and multilineage potential have led to efforts of developing clinical trials in a variety of biomedical disciplines. In general, there are two major types of stem cells, embryonic and adult stem cells.
Embryonic stem cells can be obtained from a fertilized oocyte, which are called totipotent for their capacity to produce a blastocyst that eventually could develop an embryo; or from the inner cell mass of a blastocyst. The latest ones have pluripotency capacity, meaning that they are able to specialize in cells of all three germ layers. Transplantations in vivo of embryonic stem cells are controversial for their association with tumorogenesis and the ethical and legal considerations.
Adult stem cells are self-renewing, multipotent cells localized in a wide range of adult tissues, such as bone marrow, adipose tissue, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, and spleen. The characterization of adult stem cells and the development of induced pluripotent stem cells have complicated the nomenclature and therefore when defining stem cells types’ litheness and caution is required. Mesenchymal Stem Cells (MSCs) are a type of adult stem cells widely studied and an attractive stem cell source for clinical applications [1, 3]. MSCs originate from the mesodermal germ layer and are able to encounter a multilinage differentiation. MSCs are capable of producing blood cells but these are actually derived from another cell population known as Hematopoietic stem cells (HSCs). MSCs were originally found in the stromal adherent fraction of the bone marrow sustaining the homeostatic turnover of non-haematopoietic stromal cells, regulating Hematopoietic stromal cells and contributing to vascular stability[3-4].
Due increasing interest in the therapeutic potential of the MSC and different approaches in isolation and expansion methods, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC in order to be able to compare and contrast studies outcomes. First, plastic-adherent when maintained in standard culture conditions; Second, expression of CD105, CD73 and CD90, and lack of expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules; Third, differentiate to osteoblasts, adipocytes and chondroblasts in vitro.
The development of clinical studies based on stem cells therapy and the increased demand of patients seeking for alternative and regenerative treatments, the aim of this review is to describe the history, evolution and characterization of the stem cells.
Evolution and history of stem cells
The idea of undifferentiated cells capable of regenerate and repair many types of cell populations appeared as early as 1868 with Haeckels’ concept of a stamzelle. However, it was needed many years to obtain evidence to support this theory. The first evidence of the existence of stem cells was obtained after intense research in Bone Marrow transplantation to treat the radiation sickness caused by the nuclear bombs experimentation and World War II. The incremented radioactive nuclides lead to the destruction of the bone marrow cell in exposed individuals causing anemia and leukemia. In 1963, E. A. McCulloch and J. E. Till demonstrated that a single bone marrow cell could give rise to clonal colonies of myeloid-erythroid cells in the spleen and bone marrow .
Studies in isolation and culture of stem cells started since 1964, when Kleinsmith and Pierce developed conditions to culture Embryonal Carcinoma Cells which were product of a single cell suspension from teratocarcinomas. These Embryonal Carcinoma Cells (EC cells) were able to create new neoplasm in experimental animal after injection, which suggested the presence of stem cells. By 1981, the first mouse Embryonic Stem Cell (mESC) lines were derived from the inner cell mass of a mouse embryo at the blastocyst stage and unlike EC cells, they had normal karyotypes and were able to differentiate either in vitro or in vivo[9-10]. Although, culturing of mESC was achieved the prevention of differentiation and maintenance of pluripotent mESC lines was still a difficulty. A mayor contribution to solve this problem was the addition of myeloid leukemia inhibitory factor (LIF) into the culture media to prevent differentiation and allow their growth in feeder cell-free conditions.
Although, the concept was proven, the characterization of the cells phenotype was not described and therefore, there were no protocols to isolate them until the development of flow cytometry by 1980s.
The isolation of a pure population of self-renewing pluripotent hematopoietics stem cells in mice was approached for the first time in 1988, when Spangrude et all, defined surface markers for cell separation based on monoclonal antibodies. They negatively selected surface markers characteristic of B cells (B220), granulocytes (Gr-1), myelomonocytic cells (Mac- 1), and T cells (CD4, CD8); and positively selected the cell surface differentiation antigen Thy-1low and the cell surface antigen of E13 161-7 monoclonal antibody (called stem cell antigen-I (Sca-1)). The linage depletion by the lack or presence of these surface markers lead to the enrichment of hematopoietic stem cell population in a sample; showing that as little as 30 Thy-1lowLin- (B–D–M–T–) Sca-1+ bone marrow cells were necessary to rescue one half of a group of lethally irradiated mice.
Although, the technology and knowledge to create and maintain embryonic stem cell lines was developed, cell lines derived from human ESC was first reported until 1998 due ethical issues and the availability of human embryos. Thomson et al isolated 14 inner cell masses and derived five human Embryonic Stem Cell lines from different embryos.
The development of stem cell lines became an attractive resource for generating useful somatic cells for clinical applications. However, immune rejection was still a concern. Two different approaches have been described to avoid this problem, therapeutic cloning and induced pluripotent stem cells (iPSCs). Mammalian cloning was achieved with “Dolly” in 1996 by a somatic cell nuclear transfer from a mammary gland epithelial cell to an enucleated oocyte. Although, the cloning was a success, enforcing a epigenetic plasticity on a somatic genome caused the technique to be slightly useful.
On the other hand, iPSCs technique overcomes immune rejection by reprogramming pluripotency from somatic cells. The first iPSCs were obtained from adult fibroblasts by retroviral transduction of four factors (Oct3/4, Sox2, c-Myc, and Klf4) and selected by Fbx15 expression. The resulting iPSCs showed similarities in morphology, proliferation and teratoma formation but variation in gene expression, DNA methylation patterns and failed to produce adult chimeras. The change into Nanog expression selection instead of Fbx15 expression increased embryonic stem cell like gene expression and DNA methylation patterns compared with Fbx15 iPSCs; however, 20% of the offspring developed tumours which in conjunction with other studies caused concerned for their risk of tumorigenesis[15-17]. Nowdays, there are many investigations going on to develop iPSCs without viral vectors, including Micro-RNAs and isolated proteins.
Although, the possible problems in clinical application, patient-specific iPSCs allow generation of data and investigation in what is called “disease in a dish”, for scientific, medical and pharmaceutical purposes.
A different approach for the use of Non-Embryonic Stem Cells arrived when Mesenchymal Stem Cells (MSC) were described in 1991. MSCs are multipotent cells capable of differentiating in Osteoblastocytes, Chondrocytes, Adipocytes, Dermis, Muscle, connective tissue and marrow stroma . Their multipotent activity were first observed when osteogenesis in vivo occurred after decellularized bone and bone marrow cell suspension were isotransplanted intraperitoneally to
adult mice through diffusion chambers . By the beginning of 2000s, the presence of MCSs in sources different than Bone Marrow was demonstrated; Umbilical cord, adipose tissue, amnion, placenta and deciduous teeth were shown to be a valuable source of MCSs [20-23].
MSCs are currently the stem cells with the most potential for therapeutic strategies to treat human diseases for the non-presence of ethical controversy, versatile sources and simpler manipulation for clinical applications.
Stem cell markers and characterization
Many efforts have been directed into the characterization of surface molecular markers profiles for the identification of stem cells. The fact that MSCs share common markers with other type of cells (endothelial, epithelial and muscle cells) and the variable profile of cell surface antigens depending on the source of MSCs, make it difficult to identify a general profile of markers .
The most commonly reported markers for Human Embryonic Stem Cells are Nanog, Oct-4, Sox-2, Rex-1, Dnmt3b, Lin-28, Tdgf1, FoxD3, Tert, Utf-1, Gal, Cx43, Gdf3, Gtcm1, Terf1, Terf2, Lefty A, and Lefty B . In addition, hESC are maintain as lines in laboratories and are characterized by indefinite proliferation in vitro, potential to differentiate in three embryonic germ layers, expression of specific molecular markers and maintenance of normal karyotype .
On MSCs, the most cited positive cell surface markers are CD13, CD29, CD44, CD49e, CD54, CD71, CD73, CD90, CD105, CD106, CD166, and HLA-ABC and as negative markers CD14, CD31, CD34, CD45, CD62E, CD62L, CD62P, and HLA-DR expression[27-28]. However, a difference in surface antigens depending on the source of the MSCs has been observed by the expression of STRO-1 on bone marrow-derived mesenchymal stem cells and it’s absence on adipose tissue-derived stromal cells. Although, it should be mentioned that STRO-1+ cells represent less than 5% of the bone marrow stromal population.
Hematopoietic Stem Cells have been positively selected with the use of antigen CD34 for over the past two decades. In addition, enrichment of the HSC population has been obtained by the combination of the positive markers CD34, CD133, and CD90 and CD38 as negative marker [25, 29].
Exciting developments and progress are currently being made to increase our knowledge in stem cells and open the field to more intensive clinical applications. Over 50 years of research have led us to the use of autologous adult stem cells, which are the future of regenerative medicine. This review showed the history and evolution of the stem cell research and gave an understanding of the concept and a phenotypic characterization of the different types of stem cells.
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