7-day Culture Research Articles

Toxic Effect of Streptozotocin on Cultured Mouse Hippocampal Neurons.

In primary dissociated hippocampal cell cultures from 18-day-old mouse embryos, streptozotocin in concentrations of 2-5 mM produced a dose-dependent cytotoxic effect on day 3 in vitro, whereas on day 11 of culturing, the neurons were resistant to streptozotocin. The neurons in the 3-day cultures were functionally immature, which was seen from their weak spontaneous bioelectric activity in the form of rare single action potentials; by day 11 of culturing, the neurons reached a high level of differentiation and their functional properties acquired a character of network burst activity. Thus, streptozotocin had the most pronounced cytotoxic effect on immature hippocampal neurons in vitro.

Peptide Conjugate on Multilayer Graphene Oxide Film for the Osteogenic Differentiation of Human Wharton's Jelly-Derived Mesenchymal Stem Cells.

Graphene oxide (GO) is extensively studied as a template material for mesenchymal stem cell application due to its two-dimensional nature and unique functionalization chemistries. Herein, a new type of peptide-conjugated multilayer graphene oxide (peptide/m-GO film) was fabricated and used as biomaterial for culturing human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs). The characterization of the peptide/m-GO films was performed, and the biocompatibility of the WJ-MSCs on the peptide/m-GO films was investigated. The results demonstrated that the peptide conjugate on the m-GO film did not hamper the normal growth of WJ-MSCs but supported the growth of WJ-MSCs after the 6-day culture period. In addition, the osteogenic differentiation of WJ-MSCs on the peptide/m-GO films was enhanced as compared with the parent m-GO film. Therefore, such peptide-conjugated m-GO films could provide a highly biocompatible and multifunctional 2D material to tailor the potential application of WJ-MSCs in bone tissue regeneration.

Severity of Phytophthora leaf blight disease and susceptibility of two local varieties of Colocasia to Phytophthora colocasiae Raciborski in Nsukka zone of South Eastern Nigeria

Leaf-blight disease of Colocasia caused by Phytophthora colocasiae Raciborski has been a serious impediment to cocoyam production in Nigeria. Disease severity and susceptibility of the two most cultivated local varieties “Ugwuta” (Colocasia esculenta var. antiquorum) and “Nkashi Colocasia esculenta var. esculenta) were investigated. Disease severity was visually estimated as the percentage leaf surface affected by blight, lesion or lesion-related chlorosis for each leaf of a plant using a seven-point scale of 0, 5, 10, 25, 50, 75 and 100% in three locations: Ede-Oballa, Nsukka Urban and Obukpa. Susceptibility was assessed on 2 months old potted plants of each variety inoculated with a 7-day old culture of P. colocasiae. Diameters of lesions on inoculated leaves were recorded from the 3rd - 8th day after inoculation. Data on severity were subjected to ANOVA and susceptibility of the varieties was compared with t-test. Results revealed significant LSD=4.96 (0.05) and varying degrees of leaf blight severity among varieties and locations. Variety antiquorum had significantly higher severities of 42.08, 46.40 and 47.42% at Ede-Oballa, Nsukka Urban and Obukpa respectively, compared to 34.85, 36.55 and 28.19% recorded by var. esculenta at these locations, respectively. Similarly, var. antiquorum had greater lesion diameter ranging from 0.65±0.07 cm - 3.70±0.14 cm and average diameter of 2.4±0.16cm compared to var. esculenta which had 0.41±0.14cm - 3.12±0.19 cm and average diameter of 1.80±0.16. This research has shown that varieties and locations affect the severity and susceptibility of Phytophthora leaf blight disease. This could be a guide to farmers having known that var. esculenta is less severe to Phytophthora leaf blight disease.

Engineering a 3D human intracranial aneurysm model using liquid-assisted injection molding and tuned hydrogels

Physiologically relevant intracranial aneurysm (IA) models are crucially required to facilitate testing treatment options for IA. Herein, we report the development of a new in vitro tissue-engineered platform, which recapitulates the microenvironment, structure, and cellular complexity of native human IA. A new modified liquid-assisted injection molding technique was developed to fabricate a three-dimensional hollow IA model with clinically relevant IA dimensions within a mechanically tuned Gelatin Methacryloyl (GelMA) hydrogel. An endothelium lining was created inside the IA model by culturing human umbilical vein endothelial cells over pre-cultured human brain vascular smooth muscle cells. These cellularized IA models were subjected to medium perfusion at flow rates between 6.3 and 15.75 mL/min for inducing biomimetic vessel wall shear stress (10–25 dyn/cm2) to the cells for ten days. Both cell types maintained their secretome profiles and showed more than 96% viability, demonstrating the biocompatibility of the hydrogel during perfusion cell culture at such flow rates. Based on the characterized viscoelastic properties of the GelMA hydrogel and with the aid of a fluid-structure interaction model, the capability of the IA model in predicting the response of the IA to different fluid flow profiles was mathematically shown. With physiologically relevant behavior, our developed in vitro human IA model could allow researchers to better understand the pathophysiology and treatment of IA. Statement of significanceA three-dimensional intracranial aneurysm (IA) tissue model recapitulating the microenvironment, structure, and cellular complexity of native human IA was developed. • An endothelium lining was created inside the IA model over pre-cultured human brain vascular smooth muscle cells over at least 10-day successful culture. • The cells maintained their secretome profiles, demonstrating the biocompatibility of hydrogel during a long-term perfusion cell culture. • The IA model showed its capability in predicting the response of IA to different fluid flow profiles. • The cells in the vessel region behaved differently from cells in the aneurysm region due to alteration in hemodynamic shear stress. • The IA model could allow researchers to better understand the pathophysiology and treatment options of IA.

First Report of leaf spot disease caused by Colletotrichum gloeosporioides on Chaenomeles sinensis in China.

Chaenomeles sinensis is a shrub or small arbor of the genus Chaenomeles in Rosaceae, which is widely planted in China. It is a kind of garden ornamental plant and has high economic value. Since 2020, a leaf disease occurred on the foliage of C. sinensis at the campus of Nanjing Forestry University, Nanjing, China. After investigating, C. sinensis was found with leaf spot disease at a 100% infection rate, which causing gigantic ornamental loss. Leaf spots are round to irregular distributing on the leaves, in addition, the color of spots is brown. There are yellow halos on the edge of the lesion. Small leaf tissues (3 to 4 mm2) from lesion margins were surface sterilized with 75% ethanol for 30s and then rinsed with sterile dH2O for three times. Afterwards, placed on potato dextrose agar (PDA) at 25°C. Pure cultures were obtained by monosporic isolation, and a representative isolate (NJTJ.1) was obtained. When cultured on PDA, the colony of NJTJ.1 was white and cottony. On the reverse side, the color of colony nearly light yellow. The colony were placed in the liquid Carboxymethyl cellulose (CMC) medium. After culturing for 24h in a shaker at 25℃ and 150rmp/min, the spore liquid was taken by us. The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and measured 15.1 to 23.6× 5.4 to 7.9 µm (n =30). Appressoria were one-celled, brown, thick-walled, ellipsoidal, and measured 7.7 to 13.8 × 6.4 to 10.3 µm (n =30). The morphological characteristics of NJTJ.1 fitted with the description of the Colletotrichhum gloeosporioides complex (Weir et al., 2012). For accurate identification, the internal transcribed spacer (ITS), and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS-1) were amplified with primers ITS1/ITS4, GDF/GDR, ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al, 2019), respectively. The sequences were deposited in GenBank [Accession Nos.MT984264, MW030495 and MW030496 to MW030497 for NJTJ.1]. A Blast search of GenBank showed that ITS, GAPDH, ACT and CHS-1 sequences of NJTJ.1 were 99%, 99%, 100% and 100% identical to those of C. gloeosporioides (MH571757.1 ,KY995355.1 , MN058143.1 and MN313581.1). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate NJTJ.1 clustered in the C. gloeosporioides clade with 99% bootstrap support. The pathogenicity of the NJTJ.1 was verified both on detached and living leaves. The detached leaves were inoculated with 5-mm mycelial plugs cut from the edge of 6-day old cultures on PDA and 20 μL of spore suspension (106 conidia/mL) and each treatment had 5 replicates. Controls were treated with sterile dH2O. The inocula were placed at a distance of 2 to 3 cm on the leaves which were wounded with a sterile needle. All of them were placed in 20-cm dishes on wet filter paper at 25°C. After 5 days, all the inoculated points showed lesions which were similar to those outdoor observed. Whereas, controls were asymptomatic.At the same time, the plugs of C. gloeosporioides were inoculated on living leaves.After 7 days, the leaves which were inoculated also appeared lesions. This is the first report of C. gloeosporioides causing leaf blotch on Chaenomeles sinensis in China. These data will help develop effective strategies for managing this disease.

Removal of nitrate and phosphate from simulated agricultural runoff water by Chlorella vulgaris

Microalgae such Chlorella vulgaris can effectively absorb nitrate and phosphate from contaminated water. This work characterized nitrate and phosphate removal from simulated agricultural runoff using C. vulgaris. Statistically designed experiments were used to model the following responses: (1) algal growth; (2) nitrate removal; (3) phosphate removal; (4) protein in the algal biomass; (5) chlorophyll content of the biomass; (6) the biomass phenolics content; and (7) the free radical scavenging antioxidant activity of the biomass. These response were modelled for the following key experimental factors: initial nitrate concentration in the simulated runoff (1080–3240 mg L−1, as NaNO3), initial phosphate concentration (20–60 mg L−1, as K2HPO4), photoperiod (8–24 h of light/day) and culture duration (5–15 days). The validated models were used to identify the factor levels to maximize the various responses. Nitrate removal was maximized at 85.6% when initial nitrate and phosphate concentrations were 2322 mg L−1 and 38 mg L−1 (N:P atom ratio ≈ 125:1), respectively, with a 17.2 h daily photoperiod in a 13-day culture. Phosphate removal was maximized at 95% when the initial nitrate and phosphate concentrations were 1402 mg L−1 and 56.7 mg L−1 (N:P ≈ 51:1), respectively, with a 15.7 h daily photoperiod in a 14.7-day culture. At least ~14 h of a daily photoperiod and a ~11-day culture period were required to maximize all the studied responses. C. vulgaris is edible and may be used as animal feed. Nutritional aspects of the biomass were characterized. Biomass with more than 24% protein could be produced. Under the best conditions, the chlorophyll (potential food colorants) content of the biomass was 8.5% and the maximum level of total phenolics (antioxidants) in the biomass was nearly 13 mg gallic acid equivalent g−1.

Engineering a Vascularized Hypoxic Tumor Model for Therapeutic Assessment.

Solid tumors in advanced cancer often feature a structurally and functionally abnormal vasculature through tumor angiogenesis, which contributes to cancer progression, metastasis, and therapeutic resistances. Hypoxia is considered a major driver of angiogenesis in tumor microenvironments. However, there remains a lack of in vitro models that recapitulate both the vasculature and hypoxia in the same model with physiological resemblance to the tumor microenvironment, while allowing for high-content spatiotemporal analyses for mechanistic studies and therapeutic evaluations. We have previously constructed a hypoxia microdevice that utilizes the metabolism of cancer cells to generate an oxygen gradient in the cancer cell layer as seen in solid tumor sections. Here, we have engineered a new composite microdevice-microfluidics platform that recapitulates a vascularized hypoxic tumor. Endothelial cells were seeded in a collagen channel formed by viscous fingering, to generate a rounded vascular lumen surrounding a hypoxic tumor section composed of cancer cells embedded in a 3-D hydrogel extracellular matrix. We demonstrated that the new device can be used with microscopy-based high-content analyses to track the vascular phenotypes, morphology, and sprouting into the hypoxic tumor section over a 7-day culture, as well as the response to different cancer/stromal cells. We further evaluated the integrity/leakiness of the vascular lumen in molecular delivery, and the potential of the platform to study the movement/trafficking of therapeutic immune cells. Therefore, our new platform can be used as a model for understanding tumor angiogenesis and therapeutic delivery/efficacy in vascularized hypoxic tumors.

Role of in vitro exposure to TiO2 nanoparticles in human colorectal carcinoma cells cytotoxicity

BACKGROUND AND AIM: TTitanium dioxide nanoparticles (TiO2-NPs) are a revolutionary material that are widely used in in several application fields. However, their large demand has raised many concerns about the risk of TiO2-NPs toxicity in living organisms. Although several studies showed toxic effects of TiO2-NPs on ecosystems, there are few or inconsistence evidences about the possible effects on human health of nanoparticle form (1-100 nm). The biological effect of Titanium dioxide (TiO2) on gastrointestinal tract as a possible adverse effect as been not yet clarify. In particular, the contribution of TiO2-NPs fractions to proliferative effects on tumour cells is debated. In the present study the exposure to three different Titanium chemical forms (ionic Ti, 60 nm TiO2-NPs and TiO2 food additive E171) was explored in human colorectal carcinoma cells (HCT116 and Caco-2). METHODS: Viability was assessed by MTT assay. Western Blot analysis was employed to evaluate the expression of cleaved Caspase-3 and total/cleaved PARP proteins. Colony-forming assays were performed to evaluate the cells proliferation after 7-day culture without Ti chemical forms. RESULTS:The data showed that the exposure to all three forms of Titanium decreased Caco-2 and HCT-116 cell viability in a dose dependent manner compared to untreated control after 72 hours, with a statistically significant effect starting from 1 mg/L for E171 and ionic Ti, and from 10 mg/L for TiO2-NPs. However, western blot analyses showed that the decrease of cells growth was unrelated to apoptosis. Moreover, after removing each Titanium chemical forms, cell proliferation resumed normally CONCLUSIONS:The results of the present study highlight that despite ionic Ti, TiO2-NPs and E171 affect cell viability, all the Ti chemical forms did not induce apoptosis. However, the resuming of cell proliferation after removing the particles support the notion that a better insight of the TiO2-NPs bio-effects is required, to promote a safer use practices of nanomaterials. KEYWORDS: Titanium dioxide, Nanoparticles, Apoptosis, Caco-2 cells, HCT116 cells

Effects of Hinokitiol and Dicalcium Phosphate on the Osteoconduction and Antibacterial Activity of Gelatin-Hyaluronic Acid Crosslinked Hydrogel Membrane In Vitro.

Many hydrogel-based crosslinking membranes have been designed and tailored to meet the needs of different applications. The aim of this research is to design a bifunctional hydrogel membrane with antibacterial and osteoconducting properties to guide different tissues. The membrane uses gelatin and hyaluronic acid as the main structure, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as the crosslinker, hinokitiol as the antibacterial agent, and dicalcium phosphate anhydrous (DCPA) micron particles for osteoconduction. Results show that the hydrogel membrane with added DCPA and impregnated hinokitiol has a fixation index higher than 88%. When only a small amount of DCPA is added, the tensile strength does not decrease significantly. The tensile strength decreases considerably when a large amount of modified DCPA is added. The stress–strain curve shows that the presence of a large amount of hinokitiol in hydrogel membranes results in considerably improved deformation and toughness properties. Each group impregnated with hinokitiol exhibits obvious antibacterial capabilities. Furthermore, the addition of DCPA and impregnation with hinokitiol does not exert cytotoxicity on cells in vitro, indicating that the designed amount of DCPA and hinokitiol in this study is appropriate. After a 14-day cell culture, the hydrogel membrane still maintains a good shape because the cells adhere and proliferate well, thus delaying degradation. In addition, the hydrogel containing a small amount of DCPA has the best cell mineralization effect. The developed hydrogel has a certain degree of flexibility, degradability, and bifunctionality and is superficial. It can be used in guided tissue regeneration in clinical surgery.

P–250 Mineral oil with high viscosity improves the stability of pH and osmolality in the human in vitro culture system

Abstract Study question Do commercial mineral oil brands differ in their capacity to stabilize the human embryo culture system, and is this related to the oil’s viscosity? Summary answer While the oils’ viscosity only had minor effects on temperature maintenance, it showed a direct correlation with the stability of pH and osmolality during culture. What is known already Mineral oil is a key component of the in vitro embryo culture system, which stabilizes temperature, pH and osmolality of the media during culture. Its use has been implemented worldwide for several decades and many manufacturers currently produce and commercialize oil intended for human embryo culture. Unfortunately, oil remains as one of the less characterized products in the IVF laboratory due to a lack of standardized nomenclature, production and testing. With differing physico-chemical properties, such as viscosity, oils produced by various manufacturers could behave differently to the same culture conditions and, thus, its use may need to be adjusted accordingly. Study design, size, duration Viscosity was quantified in three high-viscosity (H-V) and three low-viscosity (L-V) oils with a viscosity-meter. The required time for media’s pH to equilibrate using each oil was studied, as well as its subsequent stability outside the incubator for 30min. In-drop temperature was assessed during 15min when taking a dish outside the incubator, and again when putting it back. Additionally, each oil’s capacity to avoid media evaporation was studied with daily osmolality measurements during 7 days. Participants/materials, setting, methods pH equilibration was measured with a continuous pHmeter (Log&Guard, Vitrolife) in 4-well dishes prepared with 600µl of medium and 500µl of oil. For the other experiments, 35mm dishes with 4ml of oil and 20µl media droplets were used. pH stability was assessed after 0, 15 and 30min outside the incubator with a blood-gas-analyzer (epoc,SiemensHelthineers). A fine-gauge thermocouple was used to measure in-drop temperature loss/recovery. Daily osmolality readings were taken with a vapor pressure osmometer (Vapro5600,Wescor). Main results and the role of chance The selected oil samples had a viscosity of 115, 111, 52, 22, 18, and 12cP. The medium’s pH took approximately 12h to completely equilibrate under H-V oils, while it took less than 4h in L-V. Similarly, the rise in pH after 30min on a heated stage outside of the incubator with room atmosphere was 0.03, 0.04, 0.06, 0.13, 0.17, and 0.26, respectively. Dishes were taken out of the incubator and placed on a heated surface. In the first five minutes, the in-drop temperature loss ranged between –0.22 and –0.13oC/min, with no significant differences observed between oil types. However, temperature plateaued at a significantly higher value in L-V oils (36.5oC), compared to H-V brands (36.25–36.1oC; p = 0.0005). By contrast, all samples followed a similar pattern when the dishes were returned to the benchtop incubator, with temperature taking around 7 minutes to completely recover. Some media evaporated in all oil groups during the 7-day culture in a dry benchtop incubator. The linear regression performed to compare the evaporation rate between groups showed a statistically significant correlation between oil viscosity and the rate of evaporation (p < 0.0001), with an osmolality rise ranging between +2.55mmol/kg/day in the most viscous oil and +6.29mmol/kg/day in the least viscous. Limitations, reasons for caution While the selected oils for this study represent a wide range of options in the market, future projects could widen this selection and include additional tests, such as optimized bioassays. Results may vary between centers, and thus each laboratory should test and optimize their culture system with their own settings. Wider implications of the findings: Different oil brands have shown differing physico-chemical properties that have a direct effect on the culture system and the stability of several culture conditions. These results may be of major importance to adapt the settings and methodologies followed in each IVF laboratory according to the type of oil being used. Trial registration number Not applicable

P–115 Supplementation of healthy Sertoli cells into culture media containing follicle stimulating hormone (FSH)/testosterone (T) has no advantage in germ cell maturation

Abstract Study question In nonobstructive azoospermia (NOA) cases, whether supplementation of healthy Sertoli cells (SCs) has an effect on spermatogenic differentiation in culture medium containing FSH/T. Summary answer Expression of Crem and Acrosin increased significantly in both medium with FSH/T and medium with additional healthy SCs but there was no difference between them What is known already In NOA the induction of spermatogonial stem cells (SSCs) proliferation and differentiation has been demonstrated using different culture systems. SCs have vital roles in the regulation of spermatogenesis. Hormonal control of spermatogenesis is through FSH and T activity on SCs. Growth factors secreted by SCs via FSH, stimulate proliferation and colonization of SSCs. Although germ cells do not express androgen receptors, FSH receptors are localized on spermatogonia. It is not clear whether native SCs are sufficient for FSH/T added to the culture medium to be effective in induction of spermatogenesis, and whether supplementation of healthy SCs will increase this activity. Study design, size, duration 34 NOA and 12 obstructive azoospermia (OA) cases were included. Testicular tissue samples were taken with testicular sperm extraction (TESE) in the study and control groups. In a group of fertile cases, healthy Sertoli cells were identified and purified and then cryopreserved. Tissue samples of each case prepared in standard DMEM/F12 medium were processed in 2 separate environments containing FSH/T and FSH/T plus thawed healthy SCs for 7 days. Participants/materials, setting, methods The characterization of healthy SCs isolated from fertile cases was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. FC was used to measure the 7th day levels of specific markers expressed in spermatogonia (Vasa), meiotic cells (Crem) and post-meiotic cells (Protamine–2 and Acrosin). Main results and the role of chance In Annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT exhibited that cryopreservation didn’t inhibite the SCs proliferation compared to the pre-freezing state. Vasa and Acrosin basal levels were found to be lower in infertile patients compared to the control group (8.2% vs. 30.6% and 12.8% vs. 30.5%, p < 0.05). Compared to day 0 measurements, on the 7th day in FSH/T environment, Crem level increased by 58.8% and Acrosin level increased by 195.5% (p < 0.05). Similarly, in medium supplemented with healthy SCs, by day 7, the Crem and Acrosin levels were increased to 92.2% and 204.8%, respectively (p < 0.05). Although Vasa and Protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the 7th day increase rates of Crem, Vasa, Acrosin and Protamine–2 in either FSH/T-containing medium or in medium additionally supplemented with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8% and 232.3% vs. 198.4%, respectively, p > 0.05). Our results suggest that freezing-thawing process would not impair the viability and proliferation of SCs, and adding healthy SCs to the culture medium to correct impaired gene expression does not have an advantage over FSH/T. Limitations, reasons for caution The 7-day culture period we determined might be not sufficient for spermatogenic differentiation completion. This period could be extended in order to see further morphological differentiation may need. Wider implications of the findings: The failure of the culture media containing FSH/T to show the expected effectiveness could be thought to be due to the SCs’ inadequate response to these hormones. Therefore, healthy SCs supplementation would be needed, but this could pose ethical issues. Our findings show that it is not necessary. Trial registration number 214S532

Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets.

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

Rock inhibitor may compromise human induced pluripotent stem cells for cardiac differentiation in 3D

Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) are valuable for the understanding/treatment of the deadly heart diseases and their drug screening. However, the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging. Here, it is discovered surprisingly that Rock inhibitor (RI), used ubiquitously to improve the survival/yield of human iPSCs, induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers (i.e., ectoderm, mesoderm, and endoderm) at the commonly used concentration (10 μM). This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation. By reducing the RI to 1 μM for 3D culture, the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids. Consequently, the beating efficiency of 3D cardiac differentiation can be improved to more than 95 % in ~7 days (compared to less than ~50 % in 14 days for the 10 μM RI condition). Furthermore, the outset beating time (OBT) of all resultant cardiac spheroids (CSs) is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol (GelMA) hydrogel, showing high homogeneity (in terms of the OBT) in functional maturity of the CSs. Moreover, the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs. These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases.

First Report of Grammothele lineata Causing Stem Rot of Areca catechu in Hainan Province, China.

Areca catechu L. (areca) belongs to the Arecaceae family, which is composed of 181 genera and 2,600 species (Christenhusz and Byng 2016), is cultivated extensively in Southern and Southeastern Asia (Peng et al. 2015). Areca has a long history for its important economic and medicinal benefits and is one of the most important commercial crops in Hainan province, China. In recent years, root rot and stem rot diseases have occurred, causing areca plants to wither and even die. The serious symptoms mainly appeared in the Hainan province (Li et al. 2006). In March 2018, the rotten tissues of the diseased plants were observed to become brittle, brown, and even black from the stem base to the root; the outer leaves turned yellow, dry, and dropping in areca plantations of Qionghai county. The disease can spread from individual plants to the whole plantation in one to two years, with the characteristics of large-scale occurrence and rapid transmission, causing huge economic losses. Diseased tissues (5 × 5 mm) were disinfected with 75% ethanol for 30 s, 1% HgCl2 for 1 min, washed in sterile water, placed on potato dextrose agar (PDA) medium and incubated at 28°C (Gao et al. 2019). Pure isolates were obtained by transferring the mycelium around the diseased tissues to PDA several times. The colonies were white and cottony after culturing for 7 days. The reverse side of the colony was yellowish white. Basidiospores were hyaline, thin-walled, smooth, 1.7-1.8 x 1.6-1.7 μm (n=30) in size and circular or ellipse in shape, in addition to a dimitic hyphal system (Das et al. 2017). For molecular identification, the genomic DNA of the isolate was extracted using the thermolysis method (Zhang et al. 2010). The ribosomal internal transcribed spacer (ITS) region was amplified using the primer pairs ITS1/ITS4 (White et al. 1990), and the amplified DNA fragments were sequenced. The obtained ITS sequence (GenBank accession No. MW534416) had 99.36% identity with the reference sequence (GenBank accession No. KX013157) of Grammothele lineata Berk. & M.A. Curtis. A phylogenetic tree was constructed with software MEGA7 using the neighbor-joining method, showing that the isolate was grouped in the same clade as G. lineata. To fulfil Koch's postulates, a pathogenicity test was performed using the stems of 6-month-old healthy areca seedlings. Stem surfaces were sterilized with 70% ethanol for 30 s, rinsed three times with sterile water, and gently stabbed with a sterile needle, and then inoculated with a 1-cm-diameter colonized PDA disk from a 7-day culture on wounds, moistened with wet cotton, and wrapped with a fresh plastic wrap. Plants inoculated with sterile PDA medium plugs were used as a control. The inoculation assay was carried out twice, with five plants in both control and treatment in each test. After 20 days, the stems of the plants inoculated with the pathogen exhibited rotten symptoms, and the leaves began to become yellow and shrunken, while the control plants had only the surface of the stems discolored slightly and the inner tissue was undamaged. The fungus was re-isolated from the infected stems. Based on the morphological observations and ITS sequence analysis, the isolate was identified as G. lineata. As far as we know, this is the first report of G. lineata causing the stem rot of areca in China.

First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Spicebush in Louisiana.

In the past two decades, laurel wilt disease has significantly affected members of the Lauraceae in the southeast United States, causing widespread mortality of native redbay (Persea borbonia (L.) Spreng), and incidence of infections in avocado (Persea americana Mill.), sassafras (Sassafras albidum L.) and swamp bay (Persea palustris [Raf.] Sarg.) (Fraedrich et al., 2008, 2015, Olatinwo, et al. 2019). Laurel wilt is a vascular disease caused by Raffaelea lauricola (T.C. Harr., Fraedrich & Aghayeva), a fungus vectored by a non-native ambrosia beetle Xyleborus glabratus Eichhoff (Fraedrich et al. 2008). In August 2020, we investigated the mortality of a spicebush shrub (Lindera benzoin L.) (3.8 cm diameter at root collar, two m height) located ca. 17 mi northeast of Colfax, Grant Parish, Louisiana (31.750263° N, -92.643694° W). Evaluation of the dead shrub revealed brown, persistent foliage, and black vascular discoloration of the sapwood, typical symptoms of laurel wilt (Fig. S1). Although, beetle holes were observed on the sapwood, no beetle was found in galleries at the time. In the laboratory, a fungus consistently isolated from surface-sterilized sapwood tissues plated on potato dextrose agar (PDA) was identified as R. lauricola based on the morphological characteristics of the isolate (i.e., mucoid growth, conidiophores, and oblong/ovoid shape conidia [Harrington et al. 2008]). The fungal isolate was denoted as SB1. The identity of the fungus was confirmed by positive PCR amplification of the large subunit ribosomal RNA gene region using species-specific primers; rab-lsu-rl_F: CCCTCGCGGCGTATTATAG and rab-lsu-rl_R: GCGGGGCTCCTACTCAAA (Olatinwo, unpublished). The sequence of the isolate SB1 (GenBank Accession no. MW207371) showed 100% homology to the R. lauricola strain CBS 127349 sequence (GenBank Accession no. MH877933). The pathogenicity of SB1 on spicebush was evaluated on four healthy shrubs (average: 1 m height and 40 mm in diameter) at the location from which the original detection was made. Stems of two spicebush shrubs were inoculated with SB1 agar plugs from a 14-day old culture on PDA, while plain PDA plugs were used on the remaining two shrubs as non-inoculated controls. Agar plugs were placed in 5 mm (0.2 in) diameter hole punched on the bark with cork-borer as described by Mayfield et al (2008). After six weeks, the R. lauricola inoculated shrubs were wilted with noticeable blackened tissue discoloration in the sapwood, while the control trees remained healthy (Fig. S2). Raffaelea lauricola was re-isolated from tissue of the two inoculated, symptomatic shrubs, but not from the control trees. The sequence of the re-isolated R. lauricola isolate, denoted as SB3 (GenBank Accession no. MW207372), showed 100% homology to the R. lauricola strain CBS 127349 and isolate SB1. This first documentation of laurel wilt on spicebush in Louisiana is significant because, spicebush berries, leaves, and twigs are food sources for forest animals, birds, and insects including whitetail deer and spicebush swallowtail (Papilio troilus L.). Since its first report on sassafras in 2014 (Fraedrich et al. 2015), laurel wilt has spread across Louisiana on sassafras and swamp bay (Olatinwo et al. 2019) and has been confirmed in14 parishes. This report shows the relentless nature of the disease, as the pathogen moves from one vulnerable host to the next, expanding into new locations and threatening forest ecosystems across the southern United States.

Occurrence of Volutella Blight Caused by Pseudonectria foliicola on Boxwood in Tennessee.

Boxwood (Buxus sp. L.) is a very popular evergreen shrub in the United States which is widely used as landscape plant and fresh greenery. Boxwood 'Green velvet' (B. sinica var. insularis x B. sempervirens) plants grown in field condition exhibiting Volutella blight symptoms were found in a commercial nursery in Warren Co., Tennessee in May 2019. Leaves appeared red, brown or tan color on affected plants. Waxy, salmon pink colored fruiting bodies (sporodochia) were observed underneath the affected leaves using a hand lens (Figure 1). Leaf drop was also observed on plants. Black lesions under the bark were observed in some of the plants. The disease severity (percentage leaf area diseased) was nearly 40% and the disease incidence was nearly 30% of 1,000 plants. Infected leaf and stem tissues collected from four symptomatic plants were surface sterilized with 70% ethanol and washed with sterile distilled water. Culturing the infected leaf and stem pieces, 5-mm in size, on potato dextrose agar (PDA) consistently yielded white fluffy aerial mycelium growth with scattered salmon-color slimy masses of conidia forming from sporodochia after 10 days incubation at 25°C in a 12-h fluorescent light and dark cycle. A total of two isolates (FBG2020_396 and FBG2020_405) were hyphal tip purified on PDA. The conidia (n = 50) were hyaline, aseptate, fusiform to ellipsoidal measuring average of 7.8 × 3.3 μm (range: 4.84 to 13.2 μm × 2.2 to 4.64 μm). To confirm the pathogen identity, total DNA was extracted using UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) directly from a 5-day old culture of isolates (FBG2020_396 and FBG2020_405) on PDA. The ribosomal DNA internal transcribed spacer region (ITS), β-tubulin (tub2) and part of 28S large ribosomal subunit (LSU) regions were amplified by PCR using the primer pairs ITS 5/ITS 4, T1/BTb2 and LR0R/LR5, respectively (Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; Rehner and Samuels 1994; Vilgalys and Hester 1990; White et al. 1990). Newly generated sequences - GenBank/NCBI acc. nos. MW459251, MW465902 (ITS), MW464656, MW464657 (tub2) and MW459255, MW465903 (LSU) were 100% identical to Pseudonectria foliicola L. Lombard & Crous ex-type (CBS 123190) sequences KM231776, KM232035 and NG_058095, respectively. To complete Koch's postulates, six boxwood 'Green velvet' plants grown in 10 cm square pots (containing 40% coarse sand and 60% ground pine bark) were inoculated by spraying conidial suspension of P. foliicola [FBG2020_396 (1 × 105 conidia/mL)] obtained from 2-wk-old PDA cultures. Plants were covered with clear plastic humidity domes for 3 days and then they were maintained in a growth chamber at 25°C and 60% RH in a 12-h fluorescent light and dark cycle. Six control boxwood plants were maintained in the same environment without pathogen introduction. Pathogenicity test was conducted twice. After 10 days, typical symptoms of Volutella blight developed on the inoculated plants and microscopic examination revealed the same pathogen morphology as the original isolate. Pseudonectria foliicola was consistently re-isolated from leaves and stems. All control boxwood plants remained symptom-free and P. foliicola was not isolated from the leaves or stems. Pseudonectria foliicola causing Volutella blight has been reported on B. sempervirens in Czech Republic (Spetik et al. 2020), New Zealand (Lombard et al. 2015); Buxus sp. in Illinois, Maryland, Massachusetts, North Carolina and Washington (Salgado-Salazar et al. 2019). To our knowledge, this is the first report of Volutella blight of boxwood caused by P. foliicola in Tennessee. Pseudonectria foliicola is an opportunistic pathogen and infects weak, stressed, and injured boxwood plants/cuttings (Rivera et al. 2018). This pathogen could cause a serious economic loss to boxwood nursery growers, as it can significantly affect the ornamental value of boxwood plants and fresh greenery. Integration of sanitation practices with other disease management strategies such as biorational products and reduced-risk fungicides will be necessary for limiting the spread of pathogen and successful management of P. foliicola on boxwood in both field and postharvest conditions.

Proximate, Mineral and Phenol Content of Dioscorea Rotundata Var. Amula, Dioscorea Cayenensis Var. Igangan-Alo from Farmers’ Plots and Bounty-Fertilized, Infected with Botryodiplodia Theobromae Pat.

Fertilizer effects on infection and weight loss by Botryodiplodia theobromae Pat, was determined in white guinea yam Dioscorea rotundata variety Amula from the middle belt-region of Nigeria, and yellow guinea yam D. cayenensis variety Igangan/Alo from South West Nigeria. Tubers of both varieties were sourced from farmers’ plots from within Ogun State ( South West Nigeria) where they had been planted on three or more years previously fallowed plots, and harvested at six months after planting. Another lot of tubers of both varieties were sourced from a yam project plot at the Teaching and Research farms of the Directorate of University Farms (DUFARMS), Federal University of Agriculture (FUNAAB) Abeokuta, where they had been fertilized at the rate of 0.60 ml l-1 of Bounty fertilizer, an 8-mineral component fertilizer, and harvested also at six months after planting. Tubers of both varieties from farmers’ plots were analysed for proximate content (dry matter, moisture, ash, fat, crude protein, crude fibre and carbohydrate) phenol content and ten minerals namely Ca, N, P, K, Mg, Fe, Mn, S, Zn and B. Another set of tubers of the two varieties from farmers plots, as well as those from the Bounty-fertilized yam project plot, were inoculated with Botryodiplodia theobromae in an infection and weight loss experiment. The infection and weight loss experiment was a 2 by 2 factorial arranged in RCBD (randomized complete block design) with 4 treatment combinations and 5 replications. Tubers of both varieties from both sources inoculated with a 7-day old pure culture of B. theobromae were the four treatment combinations. Incubation period was 3 weeks. Infection in Amula (white guinea yam) was over 2500% higher than in the yellow guinea yam, Igangan. Phenol (107.89 mg/100 gdm) and calcium (16.85 mg/100 gdm) were higher in Igangan than in Amula (89.04 and 16.70 mg/100gdm respectively), and is adduced as the reason for the high infection in variety Amula. Weight loss however was 154% higher in Igangan than in Amula. Crude fibre had a negative significant correlation with weight loss (r = -0.9285, P=0.001), and crude fibre with the value 4.49% dm was higher in variety Amula than in variety Igangan (4.02%dm). This is responsible for the higher weight loss in var. Igangan. Phenol content also had a significant negative correlation with weight loss (r = -0.9586, P = 0.05), suggesting that the effect of high crude fibre in reducing weight loss outweighs that of phenol in reducing weight loss. There was high reduction of infection in the Bounty-fertilized tubers, 3.72% compared to farmers’ plot tubers which had 27.22%. Weight loss was also 0% in Bounty-fertilized tubers compared to a high 63.54% in the tubers from farmers’ plots. This indicates that the 8-mineral component fertilizer, Bounty will prolong shelf life in the white and yellow guinea yams Amula and Igangan. Five minerals namely Mg, K, Zn, Mn and B had significant correlation with weight loss (r = 0.9461, 0.9802, 0.9214, 0.9898 and 0.9122) respectively. Three of these minerals namely Zn, Mn and B content were all low with mean values of 0.25, 0.44 and 0.07 mg/100gdm respectively but Mg and K were relatively higher with mean values of 21.59 and 806.67mg/100gdm respectively. This suggests that mineral nutrition to enhance prolonged shelf life in D. rotundata var. Amula and D. cayanensis var. Igangan may be established with a balance of Mg, Zn, Mn and B in lower proportions than K, as potassium is essential in optimum yield in Dioscorea species.

Multi-Phase Ex Vivo Generation of Platelet-like Particles from CD34+ Cord Blood Cell-Derived Megakaryocytes

Currently, transfusable platelet units are solely derived from volunteer donors, but limited shelf-life and risk of infectious disease transmission present several logistical challenges for platelet storage and distribution at hospitals and blood centers. Thus, there is a growing interest in producing transfusable platelets in culture. Ex vivo platelet production is currently limited by the small quantity of input cells available for megakaryocytic (MK) differentiation, and low efficiency of generating platelets in vitro from differentiated MK cells. We present a serum-free, stroma-free, multi-phase culture process for production of CD41+/CD42+ platelet-like-particles (PLPs) from CD34+-selected umbilical cord blood (CB) cells. Our method greatly expands the number of input CD34+ cells towards the MK lineage, thus increasing the total number of CD41+/CD42+ mature MK cells and PLPs per input CD34+ cell by several fold compared to cultures without pre-expansion.CD34+ CB cells were expanded using histone deacetylase inhibitor valproic acid (VPA) and cytokines (primary culture),transferred to low oxygen conditions for further expansion and MK commitment (secondary culture), and then transferred to a final stage where MK cells mature and are fragmented under shear conditions (tertiary culture). We demonstrate that expanded pools of CD34+ CB cells differentiate into mature MK cells and produce functional CD41+/CD42+ PLPs under in vitro shear conditions.The length of the primary pre-expansion culture had a strong effect on CD41+/CD42+ presentation in subsequent culture steps. We evaluated the growth and differentiation of CD34+ cells for 16 independent CB units pre-expanded for 5 or 7 days in the presence of VPA. While 7-day VPA expansion decreased the peak percentage of CD41+/CD42+ cells in comparison to 5-day VPA expansion (E7: 21 ± 1.6% vs. E5: 30 ± 1.9% CD41+/CD42+ p=0.001), total MK cell production tended to be greater for 7-day vs. 5-day VPA expansion due to increased cell proliferation (E7: 381 ± 90 vs. E5: 290 ± 52 CD41+/CD42+ MK cells per input CD34+ cell). In comparison, unexpanded cultures produced 118 ± 29 CD41+/CD42+ cells per input CD34+ cell. Also, DNA content, as measured by percent high ploidy CD41+ cells, was positively correlated with length of pre-expansion (7-day VPA expanded: 13 ± 0.2%, 5-day VPA: 10 ± 0.5%, unexpanded: 7.1 ± 0.4% percent ≥8N CD41+ cells, p<0.001).In secondary and tertiary culture, we evaluated the effect of shear force on proliferation, MK maturation, and PLP generation. Culturing cells in an orbital shaker increased the percentage of CD41+/CD42+ cells for VPA treated cultures (shear 35 ± 2.8% vs. static 24 ± 2.6%, p = 0.02), but decreased the number of total cells in culture (shear 1270 ± 298 vs. static 2030 ± 323 total nucleated cells per CD34+ input cell). Individual MK cells within a heterogenous bulk culture were shown to release CD41+/CD42+ PLPs asynchronously over the 6-day tertiary phase. Time-course studies of PLP sampling every 24 hours revealed that 2200± 640 CD41+/CD42+ PLPs were generated per input CD34+ cell (7-day VPA expansion, n=3) at peak concentration. The CD41+/CD42+ PLP production rate of 7-day VPA cultures was greater than that for unexpanded CB CD34+ cells (1100± 120 CD41+/CD42+ PLPs per input CD34+ cell, n=3). We analyzed the morphology and functionality of generated PLPs and showed that generated PLPs became PAC-1+ and CD62P+ when activated by thrombin receptor agonists. PLPs also showed typical formations of stretched microtubule coils and actin stress fibers when spread on fibrinogen-coated glass surfaces upon activation. Interestingly, PLPs released by 7-day VPA cultures were found to be smaller than PLPs released by unexpanded and 5-day VPA cultures, and more comparable in size to donor platelets. Our study presents a proof-of-concept that the number of functional PLPs can be significantly increased via a multi-phase MK production process. DisclosuresNo relevant conflicts of interest to declare.

A Study of Novel Beta Globin Mutation Associated with Dominant Beta-Thalassemia, Aberrant Erythroid Maturation and Differentiation Compounded with Hereditary Pyropoikilocytosis

Background: Heterozygosity for a beta-thalassemia mutation is clinically insignificant; however, microcytosis and mild anemia are oftenpresent. Rare mutations in the proximal half of the coding sequence for the beta globin gene (HBB) have been shown to cause an apparent autosomal dominant beta thalassemia that is associated with more severe anemia. These mutations often produce a frame shift in HBB mRNA; however, detection of the mutant transcript and/or protein has been controversial. In genotypically identical relatives, a milder phenotype can sometimes be observed, while others have severe anemia. It has been suggested that these phenotypic differences may be mediated by variations in destruction of mutant HBB transcripts by nonsense mediated decay .Materials: We studied a Dominican man with severe microcytic hypochromic anemia without iron deficiency, suggestive of thalassemia, and his family. We performed a detailed erythrocyte evaluation, including morphologic and hemoglobinopathy evaluations, globin sequencing, hemoglobin (Hb) stability evaluation, and band-3 testing for red blood cell (RBC) membrane abnormality, and analyzed DNA for mutations of genes encoding for RBC membrane proteins. We also performed in vitro expansion of erythroid progenitors, examined erythroid differentiation and proliferation, and searched for mutant HBB transcripts.Results: The propositus was found to have a novel beta-thalassemia mutation, HBB282delT encoding a Cys93fs (frame shift). He also had Heinz bodies and was positive for an unstable Hb (by isopropanol test), but had no electrophoretically detectable mutant Hb, and had absolute reticulocytopenia (Table). Peripheral blood had a typical hereditary pyropoikilocytosis (HPP) morphology (Figure 1), confirmed by band-3 testing. Family studies (Table) showed that the situation was more complex. His asymptomatic wife and one son had sickle cell trait (Table), and two other children inherited both the HBB282delT mutation and a Hb S mutation and were positive for band-3 deficiency testing. Their RBCs had typical HPP morphology, along with sickle cells (Figure 2), and had high HbF levels (Table). Reticulocytes were not available for detection of mutant transcripts by RNA analysis; we therefore performed in vitro expansion of erythroid progenitors in a 3-week liquid erythroid culture model (Gaikwad, et al, Exp Hemat, 2007). We detected mutant HBB282delT transcripts leading to a frameshift in exon 2, codon 93, where 9 new codons downstream from the mutation were encoded. The mutant transcript was present in equal amounts with the HBB transcript from the wild allele. Analysis of erythroid proliferation and differentiation revealed a marked decrease in proliferation (as determined by number of glycophorin-positive cells) and decreased differentiation (as evaluated by temporal changes of glycophorin and transferrin receptor positivity and intensity) in the propositus and two children with HBB 282delT. Hemoglobinization of their maturing erythroid progenitors was not discernible in 14-day cultures, in contrast to his wife, the asymptomatic son and the controls who had readily discernible hemoglobinized (red) progenitors. By day 21 of culture, he and the 2 affected children had only faintly hemoglobinized cells that appeared pink rather than red, and the erythroid progenitors of the propositus had minimal differentiation. None of the affected individuals had a SPTA1 exon 2 mutation, the most common missense mutations leading to the HPP phenotype.Discussion: We describe a novel HBB mutation associated with severe anemia, reticulocytopenia, and impaired erythropoiesis with delayed erythroid maturation and impaired hemoglobinization. The presented data suggest that the mutant protein is unstable and presumably toxic to erythroid progenitors. The mutant protein is not identifiable by routine hemoglobin studies and its presence will be evaluated by studies of precipitated hemoglobin (Heinz bodies and hemoglobin precipitates after isopropanol exposure). Effects of the mutant protein on maturation and differentiation can be assessed by RNAseq of erythroid progenitors compared to normal controls of the same stage of erythroid differentiation. The molecular basis of HPP is being evaluated by DNA sequencing of targeted genes encoding RBC membrane proteins and comparing their potential functional relevance to the HPP phenotype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Insights at the Single Cell Level into the Reprogramming of Human Hematopoietic Stem Cells during Epigenetically Modified Ex Vivo Expansion

The rarity of human hematopoietic stem cells (HSCs) and the very limited ability to study these cells in vivo in the human stem cell niche are significant hurdles in understanding the regulatory networks and cues that control the balance between human HSC maintenance, survival, quiescence, self-renewal and differentiation. In murine studies, epigenetic mechanisms have been demonstrated to play important roles in regulating HSC fate (Cullen SM et al. Curr Top Dev Biol. 2014; Wang et. al. Cell Stem Cell. 2016). One key mechanism involves histone modification by acetylation, with the acetylation of histones and non-histone proteins being controlled by histone acetyltransferases and histone deacetylates (HDAC). Recent studies have demonstrated that combining histone deacetylase inhibitors (HDACi), with specific hematopoietic cytokine priming under serum-free conditions can significantly enhance the expansion ex vivo of phenotypically defined and/or NSG mouse engraftable human HSC (SCID repopulating ability cells or SRC) from cord blood (CB). Indeed, ex vivo expansion with VPA in combination with FL, TPO, SCF and IL-3 for 7 days was reported to enhance human cord blood SRCs by almost 300 fold more than cytokine priming alone and 35.9 fold more than unexpanded cord blood (Chaurasia et. al. J Clin Invest. 2014). Subsequent microarray analyses of bulk CD34+ hematopoietic progenitor cells (HSPCs) after VPA plus cytokine treatment were used to define epigenetic signatures (Gajzer et. al. Vox Sang. 2016).Given that human phenotypically-defined HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+) or long-term (LT) SRCs would be expected to increase 3-5 fold over 7 day cultures (estimated median doubling time of 36-48 hours), that LT-SRC are characterised by delayed G0 exit (with 1st division averaging 66-75h), that short-term SRC proliferate more rapidly, that HSCs are contained in both the CD90+ and CD90- subsets and that HSC develop in micro-environmental niches which provide additional regulatory cues (Laurenti E et al. Cell Stem Cell. 2015; Knapp et. al. Stem Cell Reports. 2017; Zonari E et al. Stem Cell Reports. 2017), we hypothesised that HDACi in the presence of cytokines might not only expand the CD90+ subset without differentiation but also reprogram CD90- cells to CD90+ HSCs.To test this hypothesis, we first cultured human CB CD133+ cells in SCF, FL and TPO with HDACi or vehicle addition on Nanex scaffolds (Gullo et. al. Bioinformatics. 2015). Total cell expansion over 5 days was consistently lower in HDACi cultures compared to the vehicle control, with HDACi significantly increasing the absolute numbers of phenotypically-defined HSCs. The HDACi-induced increase of HSCs was accompanied by a concomitant decrease of phenotypically defined Lin-CD38-CD34+CD45RA-CD90- cells. To confirm that cells within the CD90- population were able to give rise to CD90+ phenotypically-defined LT-HSC, we then sorted Lin-CD34+CD38-CD45RA-CD90- cells into serum-free medium containing cytokines and HDACi. After 2-day culture, the majority of CD90- cells became CD90+ culture (with no significant up-regulation of CD49f). Next, we sorted CD133+ cells, cultured for 5 days in cytokines with vehicle or HDACi, into CD90+ or CD90- subsets (Lin-CD34+CD38-CD45RA-CD49f+) and analysed their transcriptomes (100 cells per sample from 2 different donors) by RNA-sequencing using SMART-seq2 (Picelli et. al. Nature Methods. 2014). RNA-sequencing data of CD90+ cells expanded with vehicle or HDACi did not show statistically significant differences in their gene expression. However, we identified 55 genes that were differently expressed between CD90+ and CD90- (Lin-CD34+CD38-CD45RA-CD49f+) subsets but unchanged in the vehicle and HDACi conditions. Using single cell sorting and Fluidigm Biomark technology, we confirmed the differential expression of these genes between CD90+ and CD90- cells (Lin-CD34+CD38-CD45RA-CD49f+). In conclusion, we have defined gene signatures for human CD90+ and CD90- (Lin-CD34+CD38-CD45RA-CD49f+) subsets after treatment with the HDACi, and demonstrate that HDACi does not alter gene expression of the CD90+ subset but is able to both delay HSC differentiation and reprogram CD90- cells into CD90+ cells during in vitro culture. DisclosuresMead:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.