The complex formation of blood cells or hematopoiesis has been an intensely studied topic using small animals especially mouse models. Human hematopoietic stem cells (HSCs) have been studied mainly from bone marrow and from umbilical cord blood samples. HSCs are without any doubt the best-characterized adult stem cells to date. This is partly because the cell surface markers are mostly recognized in HSCs and their subsequent progenitors to study them further. HSCs are invariably important as it enable to understand the developmental dynamics and cell-fate decisions early on in any organism. Cell fate choices during normal versus leukemic transformation, can also reflect on the surface marker composition in normal HSCs versus leukemic HSCs. This identification provides immense help to examine the malformation in hematopoiesis. Especially given the fact that certain type of chronic myelocytic leukemia (CML) and acute myelocytic leukemia (AML) originate from a subset of HSCs. Interestingly, not just the blood disorders, but cardiovascular inflammatory responses may also be related to the changes in HSC differentiation potential emphasizing the significance of HSCs in many cellular functions. The safety and efficacy of HSCs in cell-based therapies underscore the enduring need to study these cells for regenerative purposes. In general, the isolation/enrichment process of stem/progenitor cells involves column-based separation or flow cytometry based sorting to obtain single or bulk cells. However, any refinement in the existing protocol, or designing a new protocol altogether will always facilitate the stem cell research field, as HSCs are assumed safe for therapeutic proposes worldwide. HSCs are mostly isolated to study their differentiation into diverse cell types by providing them with conducive signals during their culture in petri dishes. When I first encountered STEM CELLS from human umbilical cord blood for my doctoral work, it involved not only the isolation of stem cells, but also their expansion without differentiating them in culture. In this context, it was vital for us to have an optimized and simplified isolation procedure at first to get superior and adequate frequency of HSCs; particularly when aiming for in vitro expansion, it is extremely crucial.

Mouse and human HSCs differ based on the surface markers expressed by them and thus a different cocktail of antibody combination is required even though the technique behind the isolation and sorting remains pretty much the same. The current protocol that we describe here can be adapted to isolate HSCs from peripheral blood as well, especially using Ficoll based density gradient method. The important parameters that need attention is not just the process of isolation, but a) the sample quality (peripheral blood or cord blood) and how meticulously the collection process was done to avoid any incidence of contamination during downstream applications. Samples obtained without clotting as fresh as possible or within 24 hours of the collection (and processed immediately) is mandatory to get good quality HSCs being isolated from them. b) Dilution of samples always render a great separation of mononuclear cells (MNCs) from the red blood cells (RBCs) resulting an efficient separation of HSCs by either column based or flow cytometry based method. Even though experimental procedures may warrant removal of residual serum from the samples, to reduce sort stress and to maintain viability, coating the collection tube with serum is extremely advisable to keep the cells healthy and happy during the process of isolation. Understanding these factors will strengthen the efficiency of the method in procuring adequate amount of HSCs for in vitro studies and/or for in vivo transplantation procedures