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As per their findings, sequential supply of specific combinations of cytokines in a stepwise manner helped them to obtain large scale ex vivo differentiated enucleated RBCs.In first step Flt3-L, SCF, and TPO stimulated proliferation of HSCs, which was then followed by SCF, EPO, and IGF-1 aiding in the proliferation of erythroid progenitors and finally terminal erythroid differentiation was promoted by EPO and IGF-1. 2005, described an ex vivo methodology for producing fully mature human RBCs from hematopoietic stem/progenitor cells by applying G-CSF, IL-3, SCF, and EPO . demonstrated a method in which they cultured CD34 cells in a serum free medium supplemented with two cytokine sets SCF IL-3 EPO and SCF IL-3 EPO TPO Flt3 for one week, followed by coculture upon mesenchymal cells derived from cord blood for two weeks to generate an almost pure clinical grade .Erythropoietin (EPO) is a key regulator in most ex vivo protocols along with other growth factors such as SCF, IL-3, IGF-1, and Flt-3.
SCF, IL-1, IL-3, IL-6, and IL-11 stimulate pluripotent stem cell to differentiate into the CFU granulocyte, erythroid, monocyte, megakaryocyte (GEMM), and the myeloid stem cell.
The CFU-GEMM then differentiates into the specific CFU for erythroid, granulocytes, monocytes, macrophages, eosinophils, and megakaryocytes cell precursors in the presence of GM-CSF and IL3 .
Therefore, it can be assumed that ex vivo erythropoiesis can be carried out utilizing various sources, namely, embryonic stem cells (ESCs), induced pluripotent stem cells (i PSCs), and hematopoietic stem cells (HSCs), from various sources, for example, embryo, bone marrow, peripheral blood, or umbilical cord blood.
While ESCs are the most promising source but have ethical issues associated with them on the other hand, i PSCs and HSCs are next to ESCs but their isolation and maintenance increase the cost of overall ex vivo culturing process.
Neildez-Nguyen et al., 2002, in their studies demonstrated a method to amplify/expand hematopoietic stem cells (HSCs) from cord blood in a serum free culture medium.
Human nucleated erythroid cells produced by this method when injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice showed proliferation and terminal differentiated into mature enucleated red blood cells (RBCs) .
While differing in initial material and methodology used by different research groups, they all converse at a single point in using EPO as a key regulator in the ex vivo RBC generation measures.
In general, all the methods described so far mimic the marrow microenvironment through the application of cytokines and/or coculture on stromal cells, coupled with substantial amplification of stem cells with 100% terminal differentiation into fully mature, functional RBCs .
in which they used CD34 cells (from bone marrow, umbilical cord blood, and peripheral blood mobilized with G-CSF to isolate) to culture them in modified serum free media in the presence of SCF, IL-3, and erythropoietin . 2008 demonstrated a method with 100% enucleated RBCs by employing SCF, Flt-3/Flk-2 TPO in first phase followed by SCF, IL-3 EPO in the second phase . in 2011 developed a robust method to obtain ultra-high-yield of erythrocytes, with the expansion process having the capability of producing over 500 units of erythrocytes per umbilical cord blood donation using fully defined culture medium with the help of bioreactor .
As described by different research groups, all the ex vivo expansion measure would revolve around systemic and orderly use of different growth factors at various phases of ex vivo culture.