The initial use of UCB for treatment of hematopoietic disorders was based on the ability of HSCs in the UCB to reconstitute the immune system and therefore be a viable substitute for bone marrow. The HSCs are characterized by their expression of CD34, CD45, and CD133, which are hematopoietic lineage-specific antigens (McGuckin et al. 2003; Ali and Al-Mulla 2012). HSCs are not only present in UCB, but also seemed to be in a very early stage of development, as they maintain a very high proliferation rate even after over 20 years of cryopreservation (Broxmeyer et al. 2011). When McGuckin et al. (2003) examined the characteristics of UCB immature cell populations using expression of the lineage-specific antigens CD34, CD38, CD117, CD133, CD164, and Thy-1, compared to bone marrow-derived HSCs, they found that UCB contained more cells expressing an interiorCD34 (int34+) marker while being negative for exterior CD34 (ext34¯). They also determined that the UCB cells lacked exteriorized CD38 while expressing CD133. This pattern of antigen expression (intCD34⁺/extCD34¯/CD133⁺/CD38^low/−) was more highly expressed in UCB than bone marrow and indicated a more naïve phenotype. In addition to this population of very immature progenitor cells, another more primitive cell subset was also observed. These cells were quiescent stem/progenitor cells and potentially capable of long-term engraftment and ectodermal differentiation ; they expressed intCD34⁺/extCD34¯/extCD117^low (McGuckin et al. 2003)

Mesenchymal stem cells (MSCs) are also present in UCB. These cells are capable of differentiating into mesodermal cell lineages, including adipogenic, chondrogenic and osteogeneic cells (Bieback et al. 2004; Ali and Al-Mulla 2012). These cells can be identified by their lack of typical hematopoietic antigens, CD34, CD45, and CD133. Although MSCs are found in low numbers in UCB, they are highly proliferative (Ali and Al-Mulla 2012). The MSC can be isolated from the MNC fraction, forming fibroblastoid cells in culture. MSC identity was confirmed through flow cytometry by expression of CD29, CD44, CD73, and HLA-class I (Bieback et al. 2004), although there was decreased fluorescent intensity of the antigen expression compared to bone marrow-derived MSCs. These MSCs also displayed a slightly different antigen profile, with a lower expression of SH2/CD105 and a higher expression of CD106 . The isolated cells could be induced to differentiate into osteogenic, adipogenic and chondrogenic lineages, confirming their mesenchymal stem/progenitor status.

Endothelial progenitor cells (EPCs) are also found in UCB (Ingram et al. 2004). These are highly proliferative cells involved in the vascular genesis, angiogenesis and arteriogenesis. There are no specific antigens that identify all EPCs but most express CD34, CD133 and vascular endothelial growth factor receptor (VEGFR)-2 (Khakoo and Finkel 2005). Unfortunately these cells also share many surface antigens with HSCs (Asahara and Kawamoto 2004), making bioassays the only way to infer their presence. In general, these cells express endothelial surface antigens including CD31, CD105, CD141, CD144, and CD146 as well as von Willebrand Factor (vWF) but do not express CD14 and CD45.

There have been a number of studies that have compared EPCs from UCB, adult peripheral blood (APB) and bone marrow. UCB contained fifteen times more EPCs than adult peripheral blood; these cells formed larger colonies and could be ­expanded more than 100 passages without significant senescence or decreased telomere length (Ingram et al. 2004). When the CD34+ cells isolated from the UCB MNC fraction were cultured, they initially expressed CD14 and CD45 (Eggermann et al. 2003) and exhibited a monocyte-like phenotype. This expression profile ­indicates neither progenitor nor endothelial cell lineage. However, after 6 days in vitro, cells began to demonstrate endothelial phenotype, VEGFR-2+, vascular endothelial (VE)-cadherin+, and CD105+, although they remained CD34- and CD133- and CD14+ and CD45+. After about 10 days in culture, the number of cells demonstrating a endothelial-spindle shaped morphology increased .

EPCs have also been found in cryopreserved UCB (Vanneaux et al. 2010). EPCs from fresh and cryopreserved UCB were compared. Daily assessments of potential endothelial colonies were made, looking for cells with an adherent cobblestone-like morphology. The EPCs from the cryopreserved UCB contained significantly fewer endothelial colony forming cells (ECFCs) than in fresh UCB, but they had a shorter average expansion time. Even so, all cells had high CD31, CD133, VEGFR-2, and vWF expression as well as arterial and venous endothelial cell genes. EPCs from both sources were found to be capable of forming tube-like vessels and participate in in vitro wound healing, forming a cell monolayer over scar tissue. Vanneaux et al. also examined the effects of cryopreservation on cell viability and telomere length and found that fresh and cryopreserved UCB cells had no significant differences in viability or in telomere length .

Not only does UCB contain HSCs, MSCs and EPCs, there is evidence that the UCB has additional stem cell populations. Small non-hematopoietic cells have been found in UCB (McGuckin et al. 2005; Zhao et al. 2006); this population of cells expresses markers typically associated with embryonic stem cells , such as the homeobox protein NANOG, octomer-binding transcription factor-4 (OCT4), and sex determining region Y-box two (SOX2) (Ali and Al-Mulla 2012). McGuckin et al. found a population of highly proliferative embryoid-like cells from lineage-restricted immunomagnetic-separated UCB cells (McGuckin et al. 2005). These cells exhibited exponential growth for approximately 5 weeks, after an initial weeklong lag phase. Even secondary non-adherent cells, from the original UCB cell selection, once replated were capable of similarly high growth levels; these cells express some embryonic-specific antigens: OCT4, stage specific embryonic antigens three and four (SSEA-3, SSEA-4 respectively), tumor rejection antigen (TRA)-1-60, and TRA-1-81). During this study, the researchers were able to differentiate this cell type into hepatogenic cells (McGuckin et al. 2005). Another research group also isolated stem-like cells that strongly expressed embryonic markers NANOG, OCT4, SSEA-3, SSEA-4 as well as TRA-1–60 and TRA-1-81 (Zhao et al. 2006); these cells did not express markers of HSCs or MSCs. The cells also weakly expressed human leukocyte antigen (HLA) -ABC, HLA-DQ, CD40, CD80, and CD86. This is a similar immunogenic profile as embryonic stem cells , which have low immunogenicity. These cells were able to differentiate into neuronal-like cells, endothelial cells and functional pancreatic-islet cells.

UCB also has a full complement of immune cells in addition to the stem cell compartment. There are many naïve T-lymphocytes, B-lymphocytes, and monocytes in the UCB MNC. These important components of UCB likely play a large role in the low incidence of GVHD with UCB transplants (D’Arena et al. 1998). When UCB immune cells were compared to immune cells from adult peripheral blood using flow cytometry, the percentage of B cells and monocytes was similar between the two sources, but T cells were elevated in UCB.

While the early work with UCB cells focused on expansion of the cells to treat hematologic disease, the more recent focus on developing a treatment for neurologic disease or injury has required different tools. Sanchez-Ramos and associates were the first to report neural gene and protein expression from UCB-derived cells in vitro (Sanchez-Ramos et al. 2001). Using retinoic acid (RA) and nerve growth factor (NGF), they observed cellular development of UCB cells into neural-like cells that expressed glial fibrillary acidic protein (GFAP) and β-tubulin III, expressed by astrocytes and neurons, respectively. They also observed a significant increase in neural gene mRNA expression for pleiotrophin, glypican-4, neural associated growth protein 43 (GAP43), neuronal pentraxin II, and neuronal PAS1 and a significant decrease in hematopoietic genes HLA class II, attractin, macrophage receptor with collagenous structure (MARCO), leukocyte immunoglobulin-like receptor-8 (LIR-8), CD1c, erythropoietin (Epo) and its receptor. Some of the upregulated gene expression coincided with protein expression, as they also detected significant levels of Mushashi-1, β-tubulin III, pleiotrophin, and neuron-specific neural protein (NeuN) proteins. These results were confirmed and extended to describe cells with a neuronal-like morphology in addition to expression of NeuN, neurofilament, microtubule associated protein (MAP)-2 and GFAP (Ha et al. 2001; Ha et al. 2003). Later work suggested that it was CD133+ primitive stem cells present in the UCB that could produce neural cells (Jang et al. 2004). In another study, CD45- cells exposed to fibroblast growth factor (FGF)2 and human epidermal growth factor (hEGF) for 7 days could express β-tubulin III, GFAP, and galactocerebroside (Gal-C) (Bicknese et al. 2002). Similarly, lineage negative cells from UCB exposed to FGF4, SCF and flt3-ligand expressed nestin, neurofilament, A2B5 and Sox2 (Chua et al. 2009). Brain-derived neurotrophic factor (BDNF) was shown to induce neural-like differentiation of UCB MSCs (Lim et al. 2008). Based on these studies, it was a reasonable supposition that the UCB stem cells could produce neural cells and studies to develop UCB derived neural cell lines began.

One of the first attempts to develop such a cell line was reported by researchers in Poland. Bużańska et al first expanded CD34-/CD45- UCB cells in vitro and after sufficient expansion, differentiated the cells with neurobasal media containing RA and BDNF (Buzanska et al. 2002). The resulting cloned cells were positive for nestin, a marker associated with central nervous system (CNS) stem and progenitor cells. From these nestin positive cells, Bużańska et al. were able to further differentiate the UCB-derived cells with RA and BDNF into three neural progenitor cell types, expressing some neuronal and glial markers: β-tubulin III (neuronal marker), GFAP (astrocytic marker), and Gal-C, (oligodendrocytic marker). Four years later, this research group reported that they had established a neural stem cell (NSC) line from the CD34-/CD45- MNCs in UCB (Bużańska et al. 2006; Habich et al. 2006). Culturing the non-adhesive cells with different combinations of epidermal growth factor (EGF), vitamin B27, FGF2, leukemia inhibitory factor (LIF), and antibiotic antimycotic solution (AAS) with high, low or serum free media, researchers were able to establish a NSC lineage. Cells cultured with serum-free media, EGF, LIF, and FGF2 remained in an undifferentiated state as clumps of small round cells without development of an adherent basal layer. Cells cultured in low-serum, EGF, LIF and FGF2 developed an adhesive basal layer and maintained some undifferentiated free-floating cells, which expressed genes related to self-renewal (Wnt, Lif, and TGF-β pathway genes), NSCs (including Notch-2, -3, and-4), and neural commitment (Sox2). Both serum-free and low serum culture conditions yielded slower but continuous rates of expansion up to 20 days in vitro. High serum cultures yielded mostly adherent cell morphologies, and exhibited high initial growth rate, but became senescent. Researchers observed their NSC-line to be clonogenic, at a rate of about 10 %, and demonstrated neural marker expression of nestin, GFAP, and neurofilament heavy (NF-200), when cultured in low-serum media conditions. Once this cell line was developed this research group was able to induce neuronal differentiation ; the best conditions for generation of neurons were low serum and co-culture with rat astrocytes or hippocampal slices (Jurga et al. 2006). They also demonstrated that these cells expressed neurotransmitters and their receptors and were electrically active (Sun et al. 2005); unfortunately, the cells did not have the necessary sodium channels to generate an action potential. It was not clear if these cells were just not mature enough to have developed this ion channel or were not truly functional neurons.

In the first study to use UCB cells as a therapeutic for stroke , researchers intravenously (IV) administered UCB cells to rats at two time points, 24 h and 7 days post-Middle cerebral artery occlusion (MCAO) (Chen et al. 2001). They found that their treatment improved rat functional recovery and reduced neurological deficits when administered at 24 h post-MCAO. Although they did not see an infarct volume reduction post-treatment, they were able to identify UCB cells within the stroked brain mostly localized around the site of injury. These results indicated that UCB cells were capable of stimulating functional recovery post stroke, but the mechanisms by which this occurred were unclear.

Initially it was thought that the cells would act as replacement cells for necrotic tissue, especially if administered directly into the brain; it was believed that it was necessary for the cells to localize or be administered directly to the site of injury in order to ameliorate that injury, replacing necrotic cells in the infarct core. However, this view has shifted. Scientists now believe that UCB cells act indirectly through immune modulation and soluble factors (Vendrame et al. 2005). Phenomena such as angiogenesis, neurogenesis, the reduction or cessation of apoptosis and inflammation , as well as increased trophic support have all been associated with UCB cell treatment of stroke.