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Spermatogonial stem cells (SSCs) are unipotent germ cells that are at the foundation of spermatogenesis and male fertility. However, the underlying molecular mechanisms governing SSC stemness and growth properties remain elusive. We have recently identified chromodomain helicase/ATPase DNA binding protein 1-like (Chd1l) as a novel regulator for SSC survival and self-renewal, but how these functions are controlled by Chd1l remains to be resolved. Here, we applied high-throughput small RNA sequencing to uncover the microRNA (miRNA) expression profiles controlled by Chd1l and showed that the expression levels of 124 miRNA transcripts were differentially regulated by Chd1l in SSCs. KEGG pathway analysis shows that the miRNAs that are differentially expressed upon Chd1l repression are significantly enriched in the pathways associated with stem cell pluripotency and proliferation. As a proof of concept, we demonstrate that one of the most highly upregulated miRNAs, miR-486, controls SSC stemness gene expression and growth properties. The matrix metalloproteinase 2 (MMP2) gene has been identified as a novel miR-486 target gene in the context of SSC stemness gene regulation and growth properties. Data from cotransfection experiments showed that Chd1l, miR-486, and MMP2 work in concert in regulating SSC stemness gene expression and growth properties. Finally, our data also revealed that MMP2 regulates SSC stemness gene expression and growth properties through activating β-catenin signaling by cleaving N-cadherin and increasing β-catenin nuclear translocation. Our data demonstrate that Chd1l-miR-486-MMP2 is a novel regulatory axis governing SSC stemness gene expression and growth properties, offering a novel therapeutic opportunity for treating male infertility.

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the ‘omics’ technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical—failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications.

Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.

Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.

HepG2-conditioned medium (CM) facilitates early differentiation of murine embryonic stem cells (mESCs) into hematopoietic cells in two-dimensional cultures through formation of embryoid-like colonies (ELCs), bypassing embryoid body (EB) formation. We now demonstrate that three-dimensional (3D) cultures of alginate-encapsulated mESCs cultured in a rotating wall vessel bioreactor can be differentially driven toward definitive erythropoiesis and cardiomyogenesis in the absence of ELC formation. Three groups were evaluated: mESCs in maintenance medium with leukemia inhibitory factor (LIF, control) and mESCs cultured with HepG2 CM (CM1 and CM2). Control and CM1 groups were cultivated for 8 days in early differentiation medium with murine stem cell factor (mSCF) followed by 10 days in hematopoietic differentiation medium (HDM) containing human erythropoietin, m-interleukin (mIL)-3, and mSCF. CM2 cells were cultured for 18 days in HDM, bypassing early differentiation. In CM1, a fivefold expansion of hematopoietic colonies was observed at day 14, with enhancement of erythroid progenitors, hematopoietic genes (Gata-2 and SCL), erythroid genes (EKLF and β-major globin), and proteins (Gata-1 and β-globin), although ζ-globin was not expressed. In contrast, CM2 primarily produced beating colonies in standard hematopoietic colony assay and expressed early cardiomyogenic markers, anti-sarcomeric α-actinin and Gata-4. In conclusion, a scalable, automatable, integrated, 3D bioprocess for the differentiation of mESC toward definitive erythroblasts has been established. Interestingly, cardiomyogenesis was also directed in a specific protocol with HepG2 CM and hematopoietic cytokines making this platform a useful tool for the study of erythroid and cardiomyogenic development.

Embryonic stem cell (ESC) differentiation via embryoid body (EB) formation is an established method that generates the 3 germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Previously, we have enhanced mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from mESCs without standard formation of EBs. Two predifferentiation conditions were tested: (1) mESCs cultured 3 days in standard predifferentiation medium (control) and (2) mESCs cultured 3 days in HepG2 CM (CM-mESCs). Both groups were then exposed to primary differentiation for 8 days (ELC-formation period) and 14 days of hematopoietic differentiation. Enhanced mesoderm formation was observed in the CM-mESC group with an almost 5-fold increase in ELC formation (P ≤ 0.05) and higher expression of mesoderm genes-Brachyury-T, Goosecoid, and Flk-1-compared with those of control mESCs. Hematopoietic colony formation by CM-mESCs was also enhanced by 2-fold at days 7 and 14 with earlier colony commitment compared with those of control mESCs (P ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in CM-mESC culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in CM-mESC cultures. These data indicate that hematopoietic cells more quickly differentiate from CM-mESCs, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.

Abstract 276 Cord blood mononuclear cells (CBMNCs) are an ideal cell source for therapeutic applications although maintenance of fetal globin in expanded erythroid cells is problematic. We have previously developed a 3D bone marrow (BM) biomimicry through the use of a synthetic scaffold made of polyurethane (PU) coated with collagen type I which expanded CBMNCs in a cytokine-free environment for at least 28 days, with or without addition of serum. Addition of near physiological concentrations (0.2U/mL) recombinant human erythropoietin (rhuEPO) to the 3D CBMNCs serum- and cytokine-free cultures at day 7 enhanced normal erythropoiesis to enucleation (from CD45+/CD71+/CD235− to CD45−/CD71+/CD235+ stages) and promoted erythroid clonogenic capacity (CFU-E and BFU-E). However, mechanisms of erythroid expansion and globin-maturity of the cells are ill-defined. We have now extended our investigation to evaluate in situ hemoglobin and cytokine gene expression and detect cellular hemoglobin protein and cytokine production in culture supernatants. CBMNCs were separated by Ficoll-Paque density gradient centrifugation and seeded onto collagen-coated PU 3D scaffolds at 2.5×106cells/scaffold (5×5×5mm3). Cultures were established with full-medium exchange every other day in 4 conditions: 2 controls (serum-free and serum-containing cultures without rhuEPO) and serum-free cultures with addition of rhuEPO at 1.845U/mL or 0.2U/mL from day 7 onwards. Culture output was evaluated by in situ analysis and by physically extracting cells from the scaffolds. β-globin (HBB) and γ-globin (HBG) genes were detected by in situ PCR from day 0 and were consistently expressed throughout the culture period in all conditions (HBB expression was consistently higher). A decline in expression of both globins was noted at days 21 and 28 only in serum-free cultures without addition of rhuEPO. Western blot analysis of extracted cells confirmed both HBB and HBG expression in the serum-containing rhuEPO-free culture. In serum-free conditions, both HBB and HBG proteins were noted only at day 7; once rhuEPO was added to these cultures, a hemoglobin switch occurred with persistence of only HBB at days 21 and 28. Even in serum-free and rhuEPO-free cultures, HBB was present, although in lower amounts than in the rhuEPO-exposed cells, which suggested that fetal globin switching and maturation towards adult erythropoiesis occurred primarily in the serum-free cultures exposed to rhuEPO. Increased expression of GM-CSF, IL-1β and TNF was observed to day 7 and declined thereafter while IL-6 expression was observed only at day 7. There was constant high expression of TGF-β throughout the culture in all conditions, whereas TPO was not detected in any condition. The erythropoietin-receptor (EPO-R) gene was detected in all conditions, yet EPO-R was shed into the supernatant mostly in the first 7 days (maximum 42pg/ml) of serum-free cultures, declining by day 14, which corresponded to the timing of rhuEPO addition. Flt-3 was consistently detected in supernatant of the serum-containing cultures, yet declined after day 14 in serum-free cultures with and without rhuEPO. Stem cell factor (SCF), critical in early stages of erythropoiesis, was not present in culture supernatants at any time-point. Interestingly, endogenous production of EPO (maximum 0.8 mU/ml) was detected by ELISA in the first 2 weeks of both serum-containing and serum-free cultures, prior to addition of rhuEPO. High EPO concentration (maximum 2600mU/ml) was still observed in the serum-free, rhuEPO-containing cultures over the entire 28 days, suggesting that EPO was not completely utilized by the maturing cells during culture and that even lower concentrations of EPO could be beneficial. EPO detection was maximal at day 21 for serum-free cultures exposed to rhuEPO; this day 21 peak, in conjunction with the known erythroid maturation kinetics within the 3D culture, the detection of endogenous EPO production at day 14, the shedding profile of EPO-R and the hemoglobin switch, suggested that the 14–21 day time-points of culture will be important for the future study of erythroid physiology within this system. In conclusion, the 3D BM biomimicry is a good model to study erythropoiesis ex vivo, using physiological concentrations of rhuEPO and serum-free conditions rendering it suitable for future clinical applications. Disclosures: No relevant conflicts of interest to declare.

Abstract 2044 Current established protocols for the culture and differentiation of embryonic stem cells (ESCs) utilise two-dimensional (2D) tissue culture flasks/dishes. These culture methods are cumbersome and inefficient involving three stages: a) maintenance/expansion of undifferentiated ESCs, b) spontaneous differentiation through formation of embryoid bodies (EBs), and c) dissociation of EBs and replating leading to the terminal differentiation to the desired lineages. One of the major challenges in the use of ESCs for the production of red blood cells is controlling their differentiation pathway(s). Optimal culture conditions and requirements as well as precise differentiation mechanisms and cellular interactions within EBs are still not well characterised, resulting in sub-optimal control of homogenous differentiation especially due to the formation of all three germ layers. Furthermore, cavitation within EBs results in loss of available cell numbers, which reduces the yield and quality of the cellular product outcomes. To date, the most efficient protocols for the generation of oxygen-transporting, enucleated red blood cells from ESCs require co-culture with feeder cells and a multi-step process that lasts for approximately one month rendering such protocols difficult to scale-up. We have developed an integrated, single-step bioprocess that: a) uses conditioned medium (CM) derived from HepG2, a human hepatocarcinoma cell line, that stimulates mesoderm formation, b) facilitates 3D culture through encapsulation of undifferentiated mESCs in hydrogels, c) bypasses EB formation, and d) involves culture in a rotating wall vessel bioreactor that does not require passaging of the cells and is scalable and automatable. Previously, we have shown that in traditional 2D culture systems use of HepG2-CM facilitated early differentiation of mESCs into hematopoietic cells, as shown by expression of C-Myb, C-kit, Gata-2, SCL, and beta-globin genes, in comparison with that of control cultures. A significantly higher number (p≤0.001) of hematopoietic colonies was also achieved in conditioned medium-treated murine embryonic stem cells (CM)-mESC, at day 7 and 14, with a two-fold enhancement of all myeloid-erythroid progenitor colonies. Nucleated eythrocytes and macrophages were identified in the CM-mESC group as early as day 7 of culture. However, attempting to bypass the EB formation step in the 2D culture system did not produce any hematopoietic cells even by the conditioned medium-treated embryonic stem cells. Here, we now demonstrate, that single-step 3D cultures of encapsulated mESCs can produce hematopoietic cells bypassing EB formation. Specifically, undifferentiated mESCs were encapsulated (20,000 cells per hydrogel bead) and placed inside the rotating wall vessel bioreactor. The experimental group was exposed to conditioned medium supplemented with LIF for 3 days to stimulate mesoderm formation bypassing EB formation and terminal hematopoietic differentiation was accomplished by simply changing the culture medium and replacing it with 3U/ml hEPO and 40ng/ml mSCF. A significant increase in the number (p≤ 0.05) of hematopoietic colonies was observed from CM-mESCs at day 14, with a total five-fold expansion as well as enhancement of erythroid progenitors, BFU-E and CFU-E formation. A higher expression of hematopoietic genes, C-Myb, C-kit, Gata-2, SCL, as well as erythroid genes, EKLF and beta-major globin was also reported at 3 weeks of culture in low concentrations of cytokines in the bioreactor. Immunophenotypic analysis of the highly viable cells collected from the CM-mESCs group confirmed the positive expression of the proerythroblast markers (TER-119 and CD71). In conclusion, we have devised a scalable, automatable, single-step process for the derivation of mature erythrocytes from mESCs which may be used for further study of erythroid development and applications in the human system. Disclosures: No relevant conflicts of interest to declare.