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The Bioactive Peptide SL-13R Expands Human Umbilical Cord Blood Hematopoietic Stem and Progenitor Cells in Vitro

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The Bioactive Peptide SL-13R Expands Human Umbilical Cord Blood Hematopoietic Stem and Progenitor Cells in Vitro molecules Article The Bioactive Peptide SL-13R Expands Human Umbilical Cord Blood Hematopoietic Stem and Progenitor Cells In Vitro Takenobu Nii 1,2, Katsuhiro Konno 1,3 , Masaki Matsumoto 4, Kanit Bhukhai 5, Suparerk Borwornpinyo 6, Kazuhiro Sakai 7, Suradej Hongeng 8 and Daisuke Sugiyama 1,2,9,* 1 Incubation Center for Advanced Medical Science, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan; [email protected] (T.N.); [email protected] (K.K.) 2 Department of Research and Development of Next Generation Medicine, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan 3 Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan 4 Division of Cell Biology, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan; [email protected] 5 Department of Physiology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchatewi, Bangkok 10400, Thailand; [email protected] 6 Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchatewi, Bangkok 10400, Thailand; [email protected] 7 Angel Hospital, 1-11-1 Tomoda, Yahatanishiku, Kitakyushu 807-0828, Japan; [email protected] 8 Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, 270 Rama VI Road, Ratchatewi, Bangkok 10400, Thailand; [email protected] 9 Translational Research Center in Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8551, Japan Citation: Nii, T.; Konno, K.; * Correspondence: [email protected] or [email protected]; Matsumoto, M.; Bhukhai, K.; Tel.: +81-82-257-1949; Fax: +81-82-257-1993 Borwornpinyo, S.; Sakai, K.; Hongeng, S.; Sugiyama, D. The Abstract: Hematopoietic stem and progenitor cell (HSPC) transplantation is a curative treatment of Bioactive Peptide SL-13R Expands hematological disorders that has been utilized for several decades. Although umbilical cord blood Human Umbilical Cord Blood (UCB) is a promising source of HSPCs, the low dose of HSPCs in these preparations limits their Hematopoietic Stem and Progenitor Cells In Vitro. Molecules 2021, 26, 1995. use, prompting need for ex vivo HSPC expansion. To establish a more efficient method to expand https://doi.org/10.3390/molecules UCB HSPCs, we developed the bioactive peptide named SL-13R and cultured UCB HSPCs (CD34+ 26071995 cells) with SL-13R in animal component-free medium containing a cytokine cocktail. Following 9 days of culture with SL-13R, the numbers of total cells, CD34+, CD38− cells, and hematopoietic Academic Editor: Beatriz G. De la stem cell (HSC)-enriched cells were significantly increased relative to control. Transplantation of Torre cells cultured with SL-13R into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution and self-renewal ability. AHNAK, ANXA2, and PLEC all Received: 5 February 2021 interact with SL-13R. Knockdown of these genes in UCB CD34+ cells resulted in reduced numbers Accepted: 2 March 2021 of hematopoietic colonies relative to SL-13R-treated and non-knockdown controls. In summary, we Published: 1 April 2021 have identified a novel bioactive peptide SL-13R promoting expansion of UCB CD34+ cells with long-term reconstitution and self-renewal ability, suggesting its clinical use in the future. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: umbilical cord blood; hematopoietic stem/progenitor cell; peptide; cell culture published maps and institutional affil- iations. 1. Introduction Hematopoietic stem cells (HSCs) are somatic stem cells that give rise to multiple types Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. of mature and functional blood cells. HSCs have been applied to HSC transplantation This article is an open access article for more than 50 years as curative treatment for hematological disorders such as anemia distributed under the terms and and leukemia [1]. Currently, bone marrow (BM), peripheral blood (PB) and umbilical conditions of the Creative Commons cord blood (UCB) serve as cell sources for HSC transplantation. Among these, in the last Attribution (CC BY) license (https:// decades UCB has attracted increasing attention because UCB have several advantages: their creativecommons.org/licenses/by/ collection is non-invasive and safe for the donor, permissive of HLA mismatch and lower 4.0/). risk of graft-versus-host disease compared to BM or PB-derived CD34+ hematopoietic stem Molecules 2021, 26, 1995. https://doi.org/10.3390/molecules26071995 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1995 2 of 15 and progenitor cells (HSPCs) [2,3]. Nonetheless, utilization of UCB is limited as doses of HSPCs are lower in the UCB graft, which associated with inefficient engraftment and treatment-related mortality [4,5]. To overcome this limitation, investigators have established methods to expand UCB HSPCs ex vivo. Given that hematopoietic cytokines regulate HSPC function, cells are normally incubated with a cytokine cocktail during the expansion period [6,7]. Small molecules, such as nicotinamide, StemRegenin 1 (SR-1), and UM171 promote expansion of UCB HSPCs without loss of repopulating ability [8–10]. HSPCs expanded by these small molecules have been tested in a phase 1–2 trial and showed safety, feasibility, and efficacy [11–14]. During fetal development, HSCs emerge from hemogenic endothelium in the aorta- gonads-mesonephros (AGM) region [15–17], and then expand in liver [18]. Delta-like homologue 1 (DLK1) is expressed on hepatoblasts, which serve as an HSC niche and function in HSC maintenance and expansion in liver during fetal development [19–21]. Overexpression of DLK1 in hematopoietic cell inhibits differentiation [22]. The extracellular domain of DLK1 can be cleaved and give rise to a soluble fragment. This soluble DLK1 has potential to stimulate angiogenesis [23] and inhibit differentiation of adipocyte [24]. Given these findings, we hypothesized that peptide derived from the extracellular domain of DLK1 can expand HSPCs without loss of stemness. Here, we report the development of a bioactive peptide named SL-13R, the design of which was originally derived from sequence of the extracellular domain of DLK1 protein. When UCB HSPCs were cultured with SL-13R in combination with hematopoietic cytokines under animal component free conditions. SL-13R enhanced ex vivo expansion of UCB HSPCs without loss of long-term reconstitution ability. We also identified proteins that interact with SL-13R in UCB HSPCs and analyzed their potential effects on colony formation of UCB HSPCs. Overall, our work suggests an ex vivo culture method to expand UCB-derived HSPCs useful for transplantation therapy. 2. Results 2.1. SL-13R Enhances Expansion of UCB-Derived Hematopoietic Cells To evaluate the effects of the bioactive peptide, SL-13R, on proliferation, we cultured UCB HSPC with or without SL-13R using a previously described protocol [7] with modifi- cations under xeno-free conditions and then analyzed the number of live cells and HSPC population (Figure1A). After 9 days of culture, we observed a 1.4-fold increase ( p = 0.0012, n = 8) in the total number of living cells cultured with SL-13R compared to the cells cultured without SL-13R (Figure1B). Flow cytometry was used to evaluate the number of CD34+, CD38– cells and HSCs (CD34+, CD38−, CD90+, CD45RA−, CD49f+) [25] on DAY9 and observed a significantly greater number of CD34+, CD38– cells and HSCs in SL-13R-treated cells compared to control (CD34+, CD38– cells: 1.5-fold higher, p = 0.0049, n = 8; HSCs: 1.5- fold higher, p = 0.014, n = 6) (Figure1C,D). There was no significant difference in percentage of live cells (Figure S1). These effects were also observed with peripheral blood CD34+ cells cultured for 9 days with SL-13R (Figure S2A: p = 0.0048, Figure S2B: p = 0.60). To analyze the hematopoietic potential of cells cultured with SL-13R, we performed colony forming unit (CFU) assay. As shown in Figure1E, SL-13R-treated cells yielded a significantly greater number of granulocyte and macrophage (GM), erythroid, and total colony forming unit compared to control. Molecules 2021, 26, 1995 3 of 15 Figure 1. Ex vivo expansion of human UCB HSPCs by SL-13R peptide. (A) Human UCB CD34+ cells were cultured with or without SL-13R peptide (10 µg/mL) for 9 days and analyzed (B) The number of live cells with or without SL-13R (control: n = 8, SL-13R: n = 8). (C) The number of CD34+ CD38- cells with or without SL-13R (control: n = 8, SL-13R: n = 8). (D) The number of HSCs (CD34+, CD38−, CD45RA−, CD90+, CD49f+ cells) with or without SL-13R (control: n = 6, SL-13R: n = 6). (E) The number of colony forming unit (CFU) with or without SL-13R (control: n = 3, SL-13R: n = 3). The con-trol was PBS treatment. Student t test was used to test intergroup differences. * p < 0.05, ** p < 0.01. 2.2. Ex Vivo Expanded CD34+ Cells Treated with SL-13R Possess Long-Term Reconstitution and Self-Renewal Ability To determine whether the cells expanded in the presence of SL-13R maintain long-term hematopoietic reconstitution ability, we transplanted UCB HSPC, cultured in the presence or absence of SL-13R, into immunodeficient NOD/Shi-scid/IL-2Rγ knockout (NOG) mice (Figure2A). Sixteen weeks post-transplantation, we assessed the frequency of human CD45+ cells in the recipient mouse bone marrow. Percentages of human CD45+ cells in BM were 64 ± 25.35% in SL-13R-treated conditions and 27.74 ± 24.80% in controls (n = 9, p = 0.0074) (Figure2B,C). To investigate the self-renewal ability, secondary transplantations Molecules 2021, 26, 1995 4 of 15 were performed.
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