Two-step Culture System Research Articles

ELONGATED HYPOCOTYL5-mediated light signaling promotes shoot regeneration in Arabidopsis thaliana.

Injured plant somatic tissues regenerate themselves by establishing shoot or root meristems. In Arabidopsis (Arabidopsis thaliana), a two-step culture system ensures regeneration by first promoting the acquisition of pluripotency and subsequently specifying the fate of new meristems. Although previous studies have reported the importance of phytohormones auxin and cytokinin in determining the fate of new meristems, whether and how environmental factors influence this process remains elusive. In this study, we investigated the impact of light signals on shoot regeneration using Arabidopsis hypocotyls as explants. We found that light signals promote shoot regeneration while inhibiting root formation. ELONGATED HYPOCOTYL 5 (HY5), the pivotal transcriptional factor in light signaling, plays a central role in this process by mediating the expression of key genes controlling the fate of new meristems. Specifically, HY5 directly represses root development genes and activates shoot meristem genes, leading to the establishment of shoot progenitor from pluripotent callus. We further demonstrated that the early activation of photosynthesis is critical for shoot initiation, and this is transcriptionally regulated downstream of HY5-dependent pathways. In conclusion, we uncovered the intricate molecular mechanisms by which light signals control the establishment of new meristems through the regulatory network governed by HY5, thus highlighting the influence of light signals on plant developmental plasticity.

Thrombocytopenia Associated with Myelofibrosis Is Characterized By a Reduced Proportion of Megakaryocyte (MK) Biased Hematopoietic Stem Cells, Defects in MK Maturation and Increased Levels of Pro-Inflammatory Cytokines

The clinical course of patients with advanced forms of myelofibrosis (MF) can frequently be punctuated by thrombocytopenia. Marrow biopsies from these MF patients uniformly reveal megakaryocytic (MK) hyperplasia and dysplasia irrespective of the patient's platelet count. We have performed studies to better understand the origins of MF associated thrombocytopenia. We first examined the expression of G6b-B (MK-lineage specific immunoreceptor tyrosine-based inhibition motif) which is essential in promoting MK maturation, normal platelet release, platelet size and function (Becker. Blood Adv 2022). Recent reports have indicated that genetic deletion in G6b-B and loss-of-function mutations in both murine models and man lead to severe macro-thrombocytopenia and increased bone marrow fibrosis (Becker. Blood Adv 2022; Hofmann. Blood 2018; Melhem. Eur J Hematol 2017), indicating a possible role for this receptor in the development of thrombocytopenia in MF patients. We generated populations of MKs from mononuclear cells or CD34 + from thrombocytopenic or non-thrombocytopenic MFs and healthy donors (HDs) using a two-step culture system as previously described (Mosoyan. Leukemia 2017). A progressive increase of MK maturation (CD41 +CD42 +) was observed in HDs (28.5%±7) and non-thrombocytopenic MFs (32%±13.6) with a concomitant increase of total G6B expression 27%±9 and 38%±14 respectively. By contrast, MKs generated from thrombocytopenic MFs patient cells had limited CD41 +CD42 + (7%±5) and G6B (8.7%±4.7) expression (p<0.05). In addition, morphological analysis revealed that MKs from thrombocytopenic MF patients were small with hypolobulated nuclei while the MKs generated from HDs and non-thrombocytopenic MFs were larger and contained greater numbers of lobes ( Figure 1A). Psaila and coworkers have previously reported that MF is characterized by MK biased hematopoietic stem cells (HSCs) identified by increased expression of G6B (Psaila. Mol Cell 2020). We, however, determined that a reduced proportion of CD34 +CD38 - HSCs from thrombocytopenic patients expressed G6B as compared to non-thrombocytopenic MF or HD CD34 +CD38 - cells (p<0.05) ( Figure 1B). These studies indicate a reduced proportion of MK biased HSCs in thrombocytopenic MFs which likely leads to impaired MKs maturation. We next explored if the MF associated pro-inflammatory milieu also contributes to the MF MK maturation block. Thrombocytopenic MF plasma contained higher levels of YKL-40 (chitinase-3-like-1 protein), TNFα and TPO than HD plasma. For instance, MF plasma was characterized by higher levels of YKL-40 (median141,321 pg/mL) in comparison to HDs (median 19,380 pg/mL). Furthermore, YKL-40 levels were significantly higher in thrombocytopenic (median 187,696 pg/mL; p<0.05) than non-thrombocytopenic (median 78,082 pg/mL) MFs. In addition, TNFα and TPO plasma levels were also significantly higher in plasma from thrombocytopenic than non-thrombocytopenic MFs (p<0.05). We also observed that addition of either rYKL40 or rTNFα to HD-MK cultures resulted in delayed MK maturation as assessed by a reduced proportion of cells expressing CD41 +CD42 + (from 30% to 16.5% and 6.5% respectively) and G6B (from 35.5% to 22.5% and 9.5% respectively). We have previously reported that the treatment of MFs with a protein trap for TGFβ1 led to increases in platelet numbers (Mascarenhas. Clin Cancer Res 2023). The plasma from these patients prior to treatment not only had elevated levels of TGFβ1 (median 6433,917 pg/mL) but also YKL40 (median 275,640 pg/mL). Treatment of the patients with the TGFβ protein trap reduced both TGFβ1 (median 608,087 pg/mL) and YKL-40 (median 160,733 pg/mL) plasma levels, suggesting a regulatory loop between YKL-40 and TGFβ. We further explored this loop by treating the JAK2V617F+ HEL cell line with rYKL-40 in combination with rTGFβ1, which resulted in greater expression of YKL-40 (1.6 FI) than treated cells with rYKL-40 alone. In addition, treatment of HEL cells with a pan TGFβ inhibitor reduced YKL-40 protein expression (1.55 to 0.38). These data suggest the existence of an interplay between YKL40 and TGFβ on MF-MK maturation. Overall, these experiments indicate that thrombocytopenia in MF patients is due to intrinsic abnormalities in G6B originating at the level of the HSC compartment resulting in impaired MK maturation which can be further enhanced by the actions of a number of pro-inflammatory cytokines.

Optimizing Docosahexaenoic Acid (DHA) Production by Schizochytrium sp. Grown on Waste Glycerol

The aim of this study was to optimize biomass and docosahexaenoic acid (DHA) production by Schizochytrium sp. grown on waste glycerol as an organic carbon source. Parameters having a significant effect on biomass and DHA yields were screened using the fractional Plackett–Burman design and the response surface methodology (RSM). Schizochytrium sp. growth was most significantly influenced by crude glycerin concentration in the growth medium (150 g/dm3), process temperature (27 °C), oxygen in the bioreactor (49.99% v/v), and the concentration of peptone as a source of nitrogen (9.99 g/dm3). The process parameter values identified as optimal for producing high DHA concentrations in the biomass were as follows: glycerin concentration 149.99 g/dm3, temperature 26 °C, oxygen concentration 30% (v/v), and peptone concentration 2.21 g/dm3. The dry cell weight (DCW) obtained under actual laboratory conditions was 66.69 ± 0.66 g/dm3, i.e., 1.27% lower than the predicted value. The DHA concentration obtained in the actual culture was at 17.25 ± 0.33 g/dm3, which was 3.03% lower than the predicted value. The results obtained suggest that a two-step culture system should be employed, with the first phase focused on high production of Schizochytrium sp. biomass, and the second focused on increasing DHA concentration in the cells.

Enhanced production of biomass and lipids by Euglena gracilis via co-culturing with a microalga growth-promoting bacterium, Emticicia sp. EG3

BackgroundEuglena gracilis, a unicellular flagellated microalga, is regarded as one of the most promising species as microalgal feedstock for biofuels. Its lipids (mainly wax esters) are suitable for biodiesel and jet fuel. Culture of E. gracilis using wastewater effluent will improve the economics of E. gracilis biofuel production. Enhancement of the productivity of E. gracilis biomass is critical to creating a highly efficient biofuels production system. Certain bacteria have been found to promote microalgal growth by creating a favorable microenvironment. These bacteria have been characterized as microalgae growth-promoting bacteria (MGPB). Co-culture of microalgae with MGPB might offer an effective strategy to enhance microalgal biomass production in wastewater effluent culture systems. However, no MGPB has been identified to enhance the growth of E. gracilis. The objectives of this study were, therefore, to isolate and characterize the MGPB effective for E. gracilis and to demonstrate that the isolated MGPB indeed enhances the production of biomass and lipids by E. gracilis in wastewater effluent culture system.ResultsA bacterium, Emticicia sp. EG3, which is capable of promoting the growth of microalga E. gracilis, was isolated from an E. gracilis-municipal wastewater effluent culture. Biomass production rate of E. gracilis was enhanced 3.5-fold and 3.1-fold by EG3 in the co-culture system using a medium of heat-sterilized and non-sterilized wastewater effluent, respectively, compared to growth in the same effluent culture but without EG3. Two-step culture system was examined as follows: E. gracilis was cultured with or without EG3 in wastewater effluent in the first step and was further grown in wastewater effluent in the second step. Production yields of biomass and lipids by E. gracilis were enhanced 3.2-fold and 2.9-fold, respectively, in the second step of the system in which E. gracilis was co-cultured with EG3 in the first step.ConclusionEmticicia sp. EG3 is the first MGPB for E. gracilis. Growth-promoting bacteria such as EG3 will be promising agents for enhancing E. gracilis biomass/biofuel productivities.

Early ovine preantral follicles have a potential to grow until antral stage in two-step culture system in the presence of aqueous extract of Justicia insularis.

The objective of this study was to determine whether preantral follicles cultured in vitro for 7days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α-MEM+ supplemented with 0.3mg/ml J.insularis (Step 1) for 7days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6days of culture in α-MEM++ supplemented with 0.3mg/ml J.insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p<0.05) in follicular diameter and antrum formation within 6days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p<0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri-cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6days in the presence of 0.3mg/ml J.insularis.

Differentiating mouse-induced pluripotent stem cells into male germ cells through embryoid bodies

We examined the ability of the hanging drop (HD) method to differentiate induced pluripotent stem cells into male germ cells in the presence of specific growth factors. Over an eight-month period, 130 embryoid bodies (EBs) were created from mouse-induced pluripotent stem cells (miPSCs) using the hanging drop (HD) method. EBs were cultured in HDs after formation and differentiated in a 2-step culture system, each with a specific medium composition. Immunofluorescence staining was used to estimate germ cell appearance. miPSCs were maintained on mouse embryonic fibroblast (MEF) cells until confluent. MEF cells provided a cellular matrix for the miPSCs to grow on. Once confluent, the colonies were isolated by trypsinization. HDs were formed from 25-uL droplets containing miPSCs at a concentration of 1x105 cells/ml. Two male 14-week-old B6D2F1 mice were biopsied for testicular cells. Growth factors were extracted from adult mouse testes and added to the step-one medium containing Activin A, bFGF, and KSR. After 3 days, EBs were transferred to HDs containing step-two medium composed of LIF, BMP4, BMP8b, SCF, EGF, and testicular growth factors. On day 8, EBs were treated with collagenase and stained for OCT4, VASA, and DAZL. OCT4, VASA, and DAZL expression was compared between EBs cultured in the two-stage growth factor-conditioned medium system and control EBs maintained in standard culture medium to determine the stage of spermatogenesis reached. Of the isolated cells cultured from the two-step medium system, 86% were strongly positive for OCT4. In comparison, only 54% of cells isolated from control EBs cultured without differentiating factors were positive for OCT4. In addition, EBs cultured with differentiating factors yielded 31% VASA-positive cells, whereas control EBs only yielded 1% VASA-positive cells. Furthermore, EBs cultured with differentiating factors yielded 4% DAZL-positive cells, while control EBs only yielded 1% DAZL-positive cells. The retained expression of OCT4, along with the presence of VASA-positive and DAZL-positive cells, strongly indicates that the cells reached the spermatogonia/spermatocyte cell stage. Cells that were positive for VASA and DAZL without OCT4 expression also suggest that a fraction of cells were able to progress to the advanced meiotic stages of in vitro spermatogenesis. Our attempt to use the HD method together with the two-step culture system to induce the appearance and eventual maturation of male germ cells has shed light on the mechanisms of spermatogenesis in vitro. Using pluripotent stem cells to generate germ cells may benefit men afflicted by spermatogenic failure.

Evaluation of ex vivo produced endothelial progenitor cells for autologous transplantation in primates

BackgroundAutologous transplantation of endothelial progenitor cells (EPCs) is a promising therapeutic approach in the treatment of various vascular diseases. We previously reported a two-step culture system for scalable generation of human EPCs derived from cord blood CD34+ cells ex vivo. Here, we now apply this culture system to expand and differentiate human and nonhuman primate EPCs from mobilized peripheral blood (PB) CD34+ cells for the therapeutic potential of autologous transplantation.MethodsThe human and nonhuman primate EPCs from mobilized PB CD34+ cells were cultured according to our previously reported system. The generated adherent cells were then characterized by the morphology, surface markers, nitric oxide (NO)/endothelial NO synthase (eNOS) levels and Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake/fluorescein isothiocyanate (FITC)-lectin binding actives. Furthermore, the efficacy and safety studies were performed by autologous transplantation via hepatic portal vein injection in a nonhuman primate model with acute liver sinusoidal endothelial cell injury.ResultsThe mobilized PB CD34+ cells from both human and nonhuman primate were efficiently expanded and differentiated. Over 2 × 108 adherent cells were generated from 20 mL mobilized primate PB (1.51 × 106 ± 3.39 × 105 CD34+ cells) by 36-day culture and more than 80% of the produced cells were identified as EPCs/endothelial cells (ECs). In the autologous transplant model, the injected EPC/ECs from nonhuman primate PB were scattered in the intercellular spaces of hepatocytes at the hepatic tissues 14 days post-transplantation, indicating successful migration and reconstitution in the liver structure as the functional EPCs/ECs.ConclusionsWe successfully applied our previous two-step culture system for the generation of primate EPCs from mobilized PB CD34+ cells, evaluated the phenotypes ex vivo, and transplanted autologous EPCs/ECs in a nonhuman primate model. Our study indicates that it may be possible for these ex-vivo high-efficient expanded EPCs to be used in clinical cell therapy.

Replicable Expansion and Differentiation of Neural Precursors from Adult Canine Skin.

SummaryRepopulation of brain circuits by neural precursors is a potential therapeutic strategy for neurodegenerative disorders; however, choice of cell is critical. Previously, we introduced a two-step culture system that generates a high yield of neural precursors from small samples of adult canine skin. Here, we probe their gene and protein expression profiles in comparison with dermal fibroblasts and brain-derived neural stem cells and characterize their neuronal potential. To date, we have produced >50 skin-derived neural precursor (SKN) lines. SKNs can be cultured in a highly replicable fashion and uniformly express a panel of identifying markers. Upon differentiation, they self-upregulate neural specification genes, generating neurons with basic electrophysiological functionality. This unique population of neural precursors, derived from mature skin, overcomes many of the practical issues that have limited clinical translation of alternative cell types. Easily accessible, neuronally committed, and patient specific, SKNs may have potential for the treatment of brain disorders.

88 EFFECT OF BICARBONATE/CO2 LEVEL DURING EMBRYO CULTURE ON EQUINE BLASTOCYST RATE AFTER INTRACYTOPLASMIC SPERM INJECTION

Equine embryos appear to require a high glucose concentration for development to the blastocyst stage. The complete cell-culture medium, DMEM/F-12 (DM), which contains 17 mM glucose, has been widely used for equine embryo culture; however, in other species, high glucose during the early stages of embryo development is detrimental. To avoid this, we initiated a 2-step system using a low-glucose human embryo culture medium (Global) from Days 0 to 5 [Day 0 = day of intracytoplasmic sperm injection (ICSI)], with glucose (20 mM) added to the medium in the second step (Days 5 to 10; Choi et al. 2015 Reproduction 150, 31–41). We noted a high pregnancy loss rate (20%) in our clinical ICSI program (Hinrichs et al. 2014 J. Equine Vet. Sci. 34, 176), which used this 2-step Global system. Limited data are available on pregnancy with DM-produced embryos, but in one study, the loss rate was 1/13 (7.7%; Choi et al. 2011 Reproduction 142, 529–538). It is possible that use of DM in the second step of culture would better support normal blastocyst development than does Global with added glucose. However, DM is typically used at 5% CO2, and Global at 6% CO2, so use of both media would necessitate 2 sets of incubators. In the present study, we explored the use of DM in the second step of a two-step equine embryo culture system, under different CO2 environments. Oocytes were collected from research mares via follicle aspiration and were held overnight before being matured in vitro for 30 h. All media included 10% fetal bovine serum. On Days 0 to 5 after ICSI, all embryos were cultured in Global under 6% CO2 in mixed gas (5% O2 and remainder N2) at 38.2°C. In Experiment 1, on Day 5, embryos were transferred to DM prepared according to our standard method, with 14.3 mM NaHCO3 and 5 mM NaOH, and were cultured in mixed gas at either 5% CO2 or 6% CO2. Five replicates were performed. In Experiment 2, DM was prepared by our standard method, or with 24.2 mM bicarbonate and no NaOH. When pH was measured using a pH meter after media were equilibrated overnight, this higher bicarbonate provided the same pH at 6% CO2 (pH 7.3), as was achieved with the standard DM preparation at 5% CO2. Six replicates were performed. In both experiments, blastocyst development was assessed on Days 7 to 10, and blastocyst rates were compared between treatments by Fisher’s exact test. In Experiment 1, blastocyst rates were 43%, 13/30 and 27%, 8/30 for the standard DM preparation in 5% and 6% CO2, respectively (P &gt; 0.05). In Experiment 2, the blastocyst rates were 34%, 14/44 for the standard DM preparation at 5% CO2 and 43%, 19/44 for the high-bicarbonate DM at 6% CO2 (P &gt; 0.05). We conclude that a 2-step Global-DM system can support equine blastocyst production under a consistent CO2 environment (6%) if DM bicarbonate levels are adjusted to balance the increased CO2. This work was supported by the Clinical Equine ICSI Program, Texas A&amp;M University, and by the Link Equine Research Endowment Fund, Texas A&amp;M University.

Preclinical Development of a Cryopreservable Megakaryocyte Cell Product from Cord Blood Derived Hematopoietic Stem Cells

Platelet (PTL) transfusions are currently the most effective treatment for patients with thrombocytopenia. Demand for PTL transfusions has steadily increased in recent years, straining a PTL supply that is already limited due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. This dilemma has stimulated the search for alternative approaches for generating PTLs ex vivo from different sources of hematopoietic stem cells (HSCs). Although PTLs have been successfully generated in cultures initiated with primary human CD34+ cells and pluripotent stem cells, the generation of a clinically relevant PTL product ex vivo faces significant obstacles due to scalability, reproducibility and shelf life. We propose an alternative approach to overcome such obstacles by developing a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTL in vivo after transfusion into patients.Umbilical cord blood units (CBU) are FDA-approved, readily available sources for allogeneic HSC for transplantation in patients with various blood disorders. Our method utilizes a previously developed two-step culture system of megakaryopoiesis from CB CD34+ cells to generate an MK culture composed of defined MK populations: CD34+/CD41+/CD42b- MK precursors (MKP), immature CD34-/CD41+/CD42b- MK (iMK) and mature CD34-/CD41+/CD42b+ MK (mMK). While robust, the yield of MKs obtained in these cultures is restricted due to limited numbers of HSCs in CB. Our group has recently demonstrated that the numbers of CB CD34+ can be significantly expanded by epigenetic reprogramming following treatment with valproic acid (VPA). Here, we report the integration and optimization of HSC expansion with MK differentiation in order to generate a clinically relevant MK cell product. We tested 20 different culture conditions in which CD34+ cells were cultured for 5 to 8 days in the absence or presence of VPA in serum-free media with various cytokines to allow for HSC expansion. The resulting HSC pool is cultured for additional 4 to 7 days in MK differentiation/maturation media. The overall yield of CD41+ MKs obtained ranged from 8 to 33 MK per input CD34+ cell expanded in the presence of cytokines alone (n=10; mean 19.8 MK) and from 9 to 34 MK per input CD34+ cell expanded in the presence of cytokines plus VPA (n=10; mean 20.7 MK). Given that up to 2x106 CD34+ cells can be isolated from one CBU, it is anticipated that a culture yielding 28 or more MK per one CD34+ cell would generate over 56x106 MK or the equivalent of 7x105 CD41+ MK/kg/body weight for infusion into an 80 kg recipient. The culture conditions resulting in a yield of 28 or more MK per one CD34+ cell input are currently optimized to further maximize the fraction of MK generated which currently varies between 15-57% of culture. The predominant sub-population of MK resulted in these conditions consists of mMKs, regardless of VPA treatment. However, in the presence of VPA, the cultures contain a greater number of assayable CFU-MKs as compared to cytokines alone. Furthermore, preliminary studies suggest that transplantation of ex vivo generated MK leads to detectable human CD41+ cells into the BM and human PTL into the PB of NSG recipient mice. These results indicate that a MK cell product derived from CB HSCs expanded by VPA comprises not only mMK and iMK capable of immediate PTL release but also MKP and HPCs which are capable of sustained MK and PTL production. Another major advantage of a transfusion product composed of nucleated MKs is the possibility of storage by cryopreservation. Due to the fragility of mMK, we tested the cryopreservation of heterogeneous and purified MK cultures. Viability of cryopreserved MK cultures post-thaw was between 68.4-70% and no changes in the MK phenotype. Studies are ongoing to test the ex vivo and in vivo functionality of the cryopreserved MKs. In summary, starting with expanded CB HSC we created a cryopreservable cell product composed of different MK sub-populations within the MK hierarchy which is being developed into a clinically relevant therapy for treatment of thrombocytopenia. DisclosuresNo relevant conflicts of interest to declare.

Direct somatic embryogenesis and plant regeneration from leaf sheath explants of mango ginger (Curcuma amada Roxb.)

An efficient and simple procedure was systematically developed for inducing direct somatic embryogenesis and plantlet regeneration from leaf sheath explants of Curcuma amada Roxb. A two-step culture system was used to induce somatic embryogenesis. The optimized procedure resulted in direct somatic embryogenesis from 93.3% explants after 3-wk culture. Leaf sheath explants were incubated for 2 wk on medium containing 2.24 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine to initiate direct somatic embryogenesis. Thereafter, these explants were transferred to a medium containing 9.10 μM thidiazuron and 1.33 μM α-naphthaleneacetic acid. Elongated somatic embryos obtained from these cultures germinated readily, and the optimal frequency of plantlet development (86.7%) was achieved when embryos were cultured in darkness on 1/2 strength Murashige and Skoog medium containing 1.44 μM gibberellic acid. Histological and scanning electron microscopic studies showed that the initial cell divisions that led to embryo formation occurred in epidermal and subepidermal cells, followed by the development of globular and elongated structures that appeared to be somatic embryos. The presence of a clear protoderm in the globular structures and procambial strands in the elongated structures confirmed that these structures were true somatic embryos. Plantlets derived from somatic embryos were acclimatized successfully to ex vitro conditions at a survival rate of 87.43% and developed with normal phenotypes.

An optimized procedure for plant recovery from somatic embryos significantly facilitates the genetic improvement of Vitis.

Plant regeneration from grapevine (Vitis spp.) via somatic embryogenesis typically is poor. Recovery of plants from Vitis rotundifolia Michx. (muscadine grape) is particularly problematic due to extremely low efficiency, including extended culture durations required for embryo–plant conversion. Poor plant recovery is an obstacle to the selection of improved genetically modified lines. Somatic embryos (SEs) of V. rotundifolia cultivar Delicious (Del-HS) and Vitis vinifera L cultivar Thompson Seedless (TS) were used to identify culture media and conditions that promoted embryo differentiation and plant conversion; this resulted in a two-step culture system. In comparative culture experiments, C2D medium containing 6% sucrose was the most effective, among four distinct formulae tested, for inducing precocious SE germination and cell differentiation. This medium, further supplemented with 4 µM 6-benzylaminopurine (C2D4B), was subsequently determined to enhance post-germinative growth of SE. MS medium supplemented with 0.5 µM 1-naphthaleneacetic acid (MSN) was then utilized to stimulate root and shoot growth of germinated SE. An average of 35% and 80% ‘Del-HS’ and ‘TS’ SE, respectively, developed into plants. All plants developed robust root and shoot systems and exhibited excellent survival following transfer to soil. Over 150 plants of ‘Del-HS’ were regenerated and established within 2.5 months, which is a dramatic reduction from the 6- to 12-month time period previously required. Similarly, 88 ‘TS’ plant lines were obtained within the same time period. Subsequently, seven out of eight Vitis cultivars exhibited significantly increased plant conversion percentages, demonstrating broad application of the two-step culture system to produce the large numbers of independent plant lines needed for selection of desired traits.

Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis

The tumor suppressor p53 is thought to play a role in megakaryocyte (MK) development. To assess the influence of the p53 regulatory pathway further, we studied the effect of RG7112, a small molecule MDM2 antagonist that activates p53 by preventing its interaction with MDM2, on normal megakaryocytopoiesis and platelet production. This drug has been previously been evaluated in clinical trials of cancer patients where thrombocytopenia was one of the major dose-limiting toxicities. In this study, we demonstrated that administration of RG7112 in vivo in rats and monkeys results in thrombocytopenia. In addition, we identified two distinct mechanisms by which RG7112-mediated activation of p53 affected human megakaryocytopoiesis and platelet production in vitro. RG7112 promoted apoptosis of MK progenitor cells, resulting in a reduction of their numbers and RG7112 affected mature MK by blocking DNA synthesis during endomitosis and impairing platelet production. Together, the disruption of these events provides an explanation for RG7112-induced thrombocytopenia and insight into the role of the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis.

Enhancing somatic embryogenesis in avocado (Persea americana Mill.) using a two-step culture system and including glutamine in the culture medium

The development of a more efficient in vitro regeneration system for somatic embryos (SEs) of avocado (Persea americana) would facilitate the development of new superior cultivars for this valuable horticultural crop. In this study, we report a new and efficient method for maintenance and regeneration of avocado SEs. Avocado SEs of four cultivars remained healthy and viable in vitro for 11 months on a medium used for mango somatic embryogenesis, compared with 3–4 months on Murashige and Skoog medium. Various supplements and media modifications were investigated to improve the low conversion rate of regenerated plants from avocado SEs reported previously. The one-step system for regeneration of white-opaque somatic embryos (WOSEs) used solid medium only over a period of 12–14 weeks (subculturing every 6 weeks). Addition of proline and glutamine improved the total regeneration from 0 to 17.5% and 10.5%, and plant/shoot recovery from 0 to 12.5% and 5%, respectively. A two-step culture system involving the transfer of WOSEs of cultivar ‘Reed’ after 6 weeks on solid to liquid medium for 12–15 days as an intermediate step, followed by subculturing again onto solid medium for 6 weeks improved total regeneration to 29% and plant/shoot recovery to 18.3 from 0% when regenerated by subculturing on solid medium only. Supplementation with proline in the solid as well as liquid medium in the two-step culture system at 0.4g/L increased total regeneration to 35% and plant/shoot recovery to 20%. We were able to achieve highest regeneration using glutamine at 1g/L in the two-step culture system in terms of both total regeneration (58.3%, including 43.3% bipolar regeneration) and plant/shoot recovery (36.7%) rates, which were significantly higher than in any other treatment investigated.

Human preimplantation embryo development in vitro: a morphological assessment of sibling zygotes cultured in a single medium or in sequential media

A comparison was made of the development of human zygotes in either a one-step (Global® medium) or two-step culture system (Quinn's Advantage®). A total of 257 normally fertilized 2PN zygotes from 28 patients were used in the study. The study was broken down into two parts: the first concerned the development of embryos from Days 1 to 3 in Global® medium and Quinn's Advantage® cleavage medium; the second consisted of a comparison of the development of embryos from Day 3 to 5/6 in Global® medium and Quinn's Advantage® blastocyst medium. There were no significant differences between the two culture media with respect to embryo quality throughout the preimplantation phase of human embryo development as determined by the extent and variability of the cell counts, fragmentation, and nucleation. A difference was noted in the blastomere symmetry of Day 2 embryos in the two media, but was no longer apparent on examination of Day 3 embryos. No differences were noted in the rates of blastocyst development, inner cell mass (ICM), and trophectoderm (TE) scores in the two culture media. Finally, no significant differences were noted with either the proportion of blastocysts chosen for transfer or cryopreservation (vitrification). The findings support the view that two-step sequential media protocols are sufficient but not necessary to support the complete in vitro development of human preimplantation embryos.

Effect of phosphodiesterase type 3 inhibitor on nuclear maturation and in vitro development of ovine oocytes

The present study aims to investigate if prematuration culture (PMC) of ovine oocytes in the presence of a phosphodiesterase type 3 (PDE3) inhibitor cilostamide can improve the shortcomings of conventional in vitro maturation (IVM) system. Therefore, a two-step culture system consisting of 22 hours culture in the presence of 1, 10, and 20 μM cilostamide (PMC medium), followed by 22 hours culture in maturation medium, was designed. The effect of cilostamide on gap junction communications and nuclear status was studied. The variables assessed were chromosome organization, spindle pattern, polar body extrusion, and embryonic development. According to the results, inhibition of PDE3 could not permanently block nuclear maturation in ovine oocytes but it delayed the process of nuclear maturation. Elevation of intra-oocyte cAMP concentration could inhibit cumulus cells expansion and maintain gap junction communications between oocyte and cumulus cells. Deletion of cilostamide and refreshing maturation medium after 22 hours culture revealed that cumulus cells were completely expanded. The inhibitory effect induced by 1 μM cilostamide was reversible, and it increased the number of mature oocytes with aligned chromosomes and normal spindle. However, the inhibitory effects of 10 and 20 μM cilostamide was not fully reversible and was associated with deleterious effects on chromosome organization and spindle pattern. Investigation of embryonic development via parthenogenetic activation and in vitro fertilization revealed that the blastocyst rate of oocytes that were prematured with 1 μM cilostamide was not significantly different from oocytes that underwent conventional IVM but it was significantly reduced in oocytes that were prematured with 10 and 20 μM cilostamide. Our results provide the evidence that reduced cAMP via PDE3 is not the only mechanism that controls the progress of nuclear maturation in sheep oocytes, and that alternative or additional mechanisms may also exist.

Differentiation of hematopoietic stem cells isolated from peripheral blood to megakaryocyte

Objective: Thrombocytopenia remains a serious problem in patients treated with highdose chemotherapy and bone marrow transplantation. In recent years, infusion of ex vivo expanded megakaryocytes (Mk) progenitors into patients has been proposed as a strategy for shortening the time of platelet engraftment. The development of in vitro culture methods to obtain sufficient numbers of Mks from haematopoietic stem cells (HSC) is an important target in basic and clinical research projects. The aim of this study was to develop a two-step ex vivo expansion culture system of Mk progenitors from peripheral blood stem cells (PBSC). Methods: PBSC were harvested from three healthy adult donors. CD34+ cells were isolated and cultured in serum free media supplemented with thrombopoietin (TPO) (50 ng/ml), Interleukin 3 (IL-3) (20 ng/ml) and Interleukin 6 (IL-6) (20 ng/ml) for 12 days followed by an incubation with IL-6 (20 ng/ml) and TPO (50 ng/ml) for another 9 days. The differentiation of Mks was monitored by flow cytometry (% of CD34+/41+ cells). The morphology of the cells was studied by light, electron and fluorescence microscopy. Results: Morphological analysis of cells generated after 7 days of culture showed typical aspects of developing Mks. The percentage of CD41+ cells was higher than 70 on day 21. Conclusion: The results obtained in this study demonstrated that this two-step culture system is an effective method to obtain high rates of megakaryocytes. It is obvious that this promising method needs further development for clinical applications.

Development of an Improved Porcine Embryo Culture Medium for Cloning, Transgenesis and Embryonic Stem Cell Isolation

Work in our laboratory for more than two decades has focussed on the production of genetically modified pigs for xeno transplantion research. More recent work has focussed on the isolation of porcine embryonic stem cells to facilitate this as well as and other research applications. Central to this research has been the production of in vitro Produced (IVP) embryos. These embryos are produced using a twostep culture system based on NCSU23. This culture system which was developed by modifying energy substrate availability and concentrations and by adding non-essential and essential amino acids in a sequential manner. As a result of this work we have developed a culture system that better suits the changing metabolic needs of the pig embryo and produces embryos with relatively high developmental competence compared to the original formulation. These embryos can be used for a range of research applications including the isolation of embryonic stem cells.

Counseling patients regarding multiple cycles of elective oocyte cryopreservation: what have we found the second time around?

diameters and the E2 secretion in the 10ng/ml GDNF group and 100ng/ml GDNF group were significantly higher than that in the control group and 1ng/ml GDNF group (P 0.05). CONCLUSION: The human preantral follicles in the two-step serum-free fibrin-alginate three-dimensional culture system could maintain good viability. And it can be used as threedimensional culture system for the isolated follicles in vitro culture. 10ng/ml and 100ng/ml GDNF added in vitro culture medium can promote isolated human preantral follicular growth and development, and improve the secreted E2.

A two-step serum-free culture systems supports development of human preantral follicles in the presence of GDNF

To investigate the effects of the two-step serum-free fibrin-alginate three-dimensional culture system and glial cell line-derived neurotrophic factor (GDNF) on the in vitro growth and development of the isolated frozen-thawed human preantral follicles.