Stem cells (SCs) hold great promise for cell therapy, tissue engineering, and regenerative medicine as well as pharmaceutical and biotechnological applications. They have the capacity to self-renew and the ability to differentiate into specialized cell types depending upon their source of isolation. However, use of SCs for clinical applications requires a high quality and quantity of cells. This necessitates large-scale expansion of SCs followed by efficient and homogeneous differentiation into functional derivatives. Traditional methods for maintenance and expansion of cells rely on two-dimensional (2-D) culturing techniques using plastic culture plates and xenogenic media. These methods provide limited expansion and cells tend to lose clonal and differentiation capacity upon long-term passaging. Recently, new approaches for the expansion of SCs have emphasized three-dimensional (3-D) cell growth to mimic the in vivo environment. This review provides a comprehensive compendium of recent advancements in culturing SCs using 2-D and 3-D techniques involving spheroids, biomaterials, and bioreactors. In addition, potential challenges to achieve billion-fold expansion of cells are discussed.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton's jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Background: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). MS affects millions of people and causes a great economic and societal burden. There is no cure for MS. We used a novel approach to investigate the therapeutic potential of neural stem cells (NSCs) derived from human primitive mesenchymal stem cells (MSCs) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Methods: MSCs were differentiated into NSCs, labeled with PKH26, and injected into the tail vein of EAE mice. Neurobehavioral changes in the mice assessed the effect of transplanted cells on the disease process. The animals were sacrificed two weeks following cell transplantation to collect blood, lymphatic, and CNS tissues for analysis. Transplanted cells were tracked in various tissues by flow cytometry. Immune infiltrates were determined and characterized by H&E and immunohistochemical staining, respectively. Levels of immune regulatory cells, Treg and Th17, were analyzed by flow cytometry. Myelination was determined by Luxol fast blue staining and immunostaining. In vivo fate of transplanted cells and expression of inflammation, astrogliosis, myelination, neural, neuroprotection, and neurogenesis markers were investigated by using immunohistochemical and qRT-PCR analysis. Results: MSC-derived NSCs expressed specific neural markers, NESTIN, TUJ1, VIMENTIN, and PAX6. NSCs improved EAE symptoms more than MSCs when transplanted in EAE mice. Post-transplantation analyses also showed homing of MSCs and NSCs into the CNS with concomitant induction of an anti-inflammatory response, resulting in reducing immune infiltrates. NSCs also modulated Treg and Th17 cell levels in EAE mice comparable to healthy controls. Luxol fast blue staining showed significant improvement in myelination in treated mice. Further analysis showed that NSCs upregulated genes involved in myelination and neuroprotection but downregulated inflammatory and astrogliosis genes more significantly than MSCs. Importantly, NSCs differentiated into neural derivatives and promoted neurogenesis, possibly by modulating BDNF and FGF signaling pathways. Conclusions: NSC transplantation reversed the disease process by inducing an anti-inflammatory response and promoting myelination, neuroprotection, and neurogenesis in EAE disease animals. These promising results provide a basis for clinical studies to treat MS using NSCs derived from primitive MSCs.

Background: Efficacy and safety of anticancer drugs are traditionally studied using cancer cell lines and animal models. The thienodiazepine class of BET inhibitors, such as JQ1, has been extensively studied for the potential treatment of hematological malignancies and several small molecules belonging to this class are currently under clinical investigation. While these compounds are well known to inhibit cancer cell growth and cause apoptosis, their effects on stem cells, particularly mesenchymal stem cells (MSCs), which are important for regeneration of damaged cells and tissues, are unknown. In this study we employed umbilical cord derived MSCs as a model system to evaluate the safety of JQ1. Methods: Cord derived MSCs were treated with various doses of JQ1 and subjected to cell metabolic activity, apoptosis, and cell cycle analyses using MTT assay, Annexin-V/FITC and PI staining, and flow cytometry, respectively. The effect of JQ1 on gene expression was determined using microarray and quantitative real-time reverse transcriptase polymerase chain reaction analysis. Furthermore, protein expression of apoptotic and neuronal markers was carried out using western blot and immunostaining, respectively. Results: Our results showed that JQ1 inhibited cell growth and caused cell cycle arrest in G1 phase but did not induce apoptosis or senescence. JQ1 also down-regulated genes involved in self-renewal, cell cycle, DNA replication, and mitosis, which may have negative implications on the regenerative potential of MSCs. In addition, JQ1 interfered with signaling pathways by down regulating the expression of WNT, resulting in limiting the self-renewal. These results suggest that anticancer agents belonging to the thienodiazepine class of BET inhibitors should be carefully evaluated before their use in cancer therapy. Conclusions: This study revealed for the first time that JQ1 adversely affected MSCs, which are important for repair and regeneration. JQ1 specifically modulated signal transduction and inhibited growth as well as self-renewal. These findings suggest that perinatal MSCs could be used to supplement animal models for investigating the safety of anticancer agents and other drugs.

Human umbilical cord (hUC) blood and tissue are non-invasive sources of potential stem/progenitor cells with similar cell surface properties as bone marrow stromal cells (BMSCs). While they are limited in cord blood, they may be more abundant in hUC. However, the hUC is an anatomically complex organ and the potential of cells in various sites of the hUC has not been fully explored. We dissected the hUC into its discrete sites and isolated hUC cells from the cord placenta junction (CPJ), cord tissue (CT), and Wharton's jelly (WJ). Isolated cells displayed fibroblastoid morphology, and expressed CD29, CD44, CD73, CD90, and CD105, and showed evidence of differentiation into multiple lineages in vitro. They also expressed low levels of pluripotency genes, OCT4, NANOG, SOX2 and KLF4. Passaging markedly affected cell proliferation with concomitant decreases in the expression of pluripotency and other markers, and an increase in chondrogenic markers. Microarray analysis further revealed the differences in the gene expression of CPJ-, CT- and WJ-hUC cells. Five coding and five lncRNA genes were differentially expressed in low vs. high passage hUC cells. Only MAEL was expressed at high levels in both low and high passage CPJ-hUC cells. They displayed a greater proliferation limit and a higher degree of multi-lineage differentiation in vitro and warrant further investigation to determine their full differentiation capacity, and therapeutic and regenerative medicine potential.