On the other hand, UC-MSCs are heterogenous in terms of stemness gene and neurogenic gene expression. form secondary neurospheres and can be readily induced to become neurons and glial cells. Real-time PCR showed significantly upregulated expression of multiple stemness and neurogenic genes after induction. RNA transcriptional profiling study showed that UC-MSC-derived neurospheres had a unique transcriptional profile of their own, with features of both UC-MSCs and neural stem cells. HSPC150 RayBio human growth factor cytokine array analysis showed significantly upregulated expression levels of multiple neurogenic and angiogenic growth factors, skewing toward a neural stem cell phenotype. Thus, we believe that these UC-MSC-derived neurospheres have amenable features of both MSCs and neural stem/progenitor cells and have great potential in future stem cell transplantation clinical trials targeting neurological disorders. 1. Introduction Mesenchymal stem cells are adult stem cells derived from mesenchymal tissues. Human MSCs can be obtained from various sources such as bone marrow, umbilical cord, cord blood, adipose tissue, even the dental pulp [1C3]. They have great advantages of easy accessibility, easy manipulation, and low HLA typing restriction, VU 0361737 combined with their promising features of self-renewal and multipotency, making them the most commonly used adult stem cells in regenerative medicine. MSCs have been widely used in clinical trials for the treatment of diseases including hematological diseases, graft-versus-host diseases, diabetes, end-stage diseases in the liver, kidney, and lung, autoimmune diseases, and various neurological diseases [4C12]. There are several important limitations for current stem cell therapy trials using MSCs for the treatment of neurological diseases. First, for the treatment of neurological diseases, it would be clinically more relevant and efficient if we could use neural stem cells in these trials. But so far, human neural stem cells are extremely difficult to obtain due to ethical restrictions. Even if human neural stem cell can be obtained, the patient will need lifelong immunosuppressive agents . Human umbilical cord-derived mesenchymal stem cells express little HLA antigen and hence can be safely used in a heterologous transplant setting , but here comes the second problem. Although MSCs have been shown to be efficiently induced to form neurons under certain induction methods in ex vivo experiments [15C19], when these cells are infused in vivo when they need in vivo microenvironment cues to transdifferentiate, they do not perform so well [20, 21]. It would therefore be ideal to find a cell source which combines the strength of both MSCs and neural stem cells, having both of their desirable features in one cell source. In the present study, we reported for the first time that UC-MSCs can be efficiently induced to form neurosphere-like cells under standard culture conditions used for neurospheres (DMEM/F12, EGF, bFGF, N2, and B27) within 12 hours. These MSC-derived neurospheres can self-renew to form secondary neurospheres and can be readily induced to form neurons and glial cells. Real-time PCR showed significantly elevated expression of multiple neurogenic VU 0361737 genes including and after induction. RNA sequencing analysis revealed that these UC-MSC-derived neurospheres have a distinctive transcriptional profile, different from both MSCs and human neural stem cells. Human growth factor analysis on these MSC-derived neurospheres showed that they had greatly enhanced expression in many neurogenic and angiogenic cytokines. Therefore, these MSC-derived neurospheres represent a new source for neural stem/progenitor cells. VU 0361737 They display self-renewal and multipotentialities comparable to neural stem/progenitor cells while still maintaining a low HLA restriction profile of typical MSCs. We believe that these cells have amenable features of both MSCs and neural stem/progenitor cells and will find themselves of tremendous use in future stem cell transplantation clinical trials for various neurological diseases. 2. Methods 2.1. Ethics Statement All methods used in this study were carried out in accordance with the approved ethical guidelines of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, China. The study protocol was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan, Hubei, China. Informed consent VU 0361737 was obtained from all subjects prior to the study. 2.2. Isolation and Characterization of UC-MSCs Human umbilical cords were obtained from mothers (20C30 years old) planning on cesarean sections after obtaining written informed consent. UC-MSCs were isolated and characterized as previously described [22, 23]. For osteogenic differentiation, UC-MSCs were cultured in DMEM/F12 (Lonza, Walkersville, MD, USA) supplemented with 10% FBS, 0.1?mM dexamethasone, 10?mM values < 0.05 were considered to indicate statistical significance. values < 0.01 were considered statistically very significant. All analyses VU 0361737 were performed using GraphPad Prism 8 (San.