## 1 Introduction  
Mesenchymal stromal/stem cells (MSCs) represent clinically valuable cell populations capable of self-renewal, multipotent differentiation, immunomododulation, and great potential in tissue repair and regeneration [1]. The progress in tissue engineering and regenerative medicine has created a vast demand for the production of MSCs. This fact requires on one hand the identification of stem cell sources that promise higher yields, and on the other the development of a better protocol for the isolation, expansion, and quantification of MSCs. Birth-associated organs such as the umbilical cord (UC) and placenta are of great interest to stem cell researchers as an alternative to adult MSCs [2; 3]. These extraembryonic organs are routinely discarded following the delivery of a baby and can be obtained easily in large quantities without ethical implications [4]. Several studies have reported that MSCs isolated from the UC exhibit similar immunophenotypic characteristics to MSCs from bone marrow (known as a \"gold standard\" for MSCs) and adipose tissue [4; 5] and greater proliferative, differentiation, immunosuppressive, and regenerative potential [6; 7; 8]. Hence, the UC has become one of the most popular birth-associated sources of MSCs, being developedfor both pre-clinical and clinical trials such as graft-versus-host disease [9], premature ovarian failure [10], osteoarthritis [11; 12], psoriasis [13], and diabetes mellitus [14]. Among the other abilities of the MSCs derived from [PERSON]'s jelly (WJ) and the subamnion (SA), like great proliferation and various cell types differentiation capacity, these MSCs can also modulate the immune response and possesses low tumorigenicity. The aforementioned qualities make them suitable for the treatment of various pathologies in the bones, muscles, nerve system, reproductive system, and internal parenchymal organs, as well as diabetes, cancer, and infectious diseases [15; 16; 17]. The unique features of the WJ-MSCs and SA-MSCs allow them to be used for allogeneic and xenogeneic transplantation, thanks to their lower immunogenicity [18; 19; 20; 21]. Nonetheless, WJ-MSCs and SA-MSCs release extracellular vesicles like exosomes and microvesicles which contain various biologically active substances and can be modulated by stimuli added to the culture environment of the stem cells, which makes it even more suitable for treatment of certain medical conditions [15; 22]. Anatomically, the umbilical cord is composed of an amniotic epithelium covering the surface of the UC, followed by the subamnion that surrounds a central core of mucoid connective tissue (\"Wharton's jelly\") containing three vessels--a vein and two arteries [23; 24]. MSC populations with varied stemness characteristics have been isolated from various compartments of the UC such as the amn (amnionic epithelial membrane), subamnion region (cord lining membrane), Wharton's jelly, and perivascular areas [25; 26; 27]. The potential for proliferation, the differentiation capacity, and the expression of MSC-specific profile and pluripotency-associated genes (OCT4, SOX2, NANOG, and SSEA-4) have been examined extensively in UC-MSCs. Evidence suggests that the culture conditions and isolation technique have an impact on both the cell yield and the biological properties of MSCs [28; 29]. Moreover, the absence of standardization in UC-MSC isolation and growth affects the reproducibility and comparability of outcomes in the studies [30]. Umbilical cord cells are obtained by three ways--enzyme digestion, explant tissue approach, and combinations of these two techniques [28; 31; 32]. In the enzymatic methods, the numbers and types of proteolytic enzymes used may lead to the damage of the cell membranes and the loss of ability to adhere or to proliferate, and may also alter MSC properties by the degradation of surface receptors [28; 33]. By contrast, the explant methods employ the property of MSCs to migrate out of the tissue and to adhere to the plastic surface, avoiding cell damage [34].