Mesenchymal stem cells (MSCs) have raised immense hopes for their vast applications in regenerative medicine. It has paved its way to a large number of clinical trials for a broad range of diseases. So far, several studies have provided promising results, but more research is required for their further validation due to varying outcomes. This might be due to varying sources of cells, heterogeneity in MSCs cultures and absence of reliable protocols for isolation and enrichment of the naïve stem cell fraction. MSCs can be isolated from many tissues under different culture conditions but appropriate cell source is needed to be investigated for their efficient applications in clinical practice. Human umbilical cord blood (hUCB) is the richest source of non–embryonic stem cells. The collection of MSCs from hUCB is easier, less expensive and non–invasive. UCB–MSCs are less immunogenic, and therefore do not elicit the proliferative response of allogeneic lymphocytes in vitro and they elicit a lower incidence of graft rejection and post–transplant infections compared with other sources. Hence the present study has been taken in consideration for standardization of appropriate in vitro protocols of MSCs isolation and enrichment from hUCB .