Jungwoon Lee

Optimal pH control of batch processes for production of curdlan by Agrobacterium species.

We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species. The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin. The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters. The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion. By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L−1) compared to that of constant pH operation (36 g L−1).

Influence of agitation speed on production of curdlan by Agrobacterium species

Abstract Influence of dissolved oxygen level on production of curdlan by Agrobacterium species was investigated. Preliminary shake flask experiments showed that both cell growth and curdlan production were higher at a smaller volume of medium (50–100 ml in 500 ml flasks). As culture volume increased from 100 ml to 300 ml, both cell concentration and curdlan production decreased, indicating that higher oxygen transfer is required for a higher production of curdlan. Time profiles of cell concentration and curdlan production in a 5-liter jar fermentation at different agitation speeds, ranging from 300 rpm to 700 rpm, supported the fact of higher production of curdlan at higher oxygen transfer rate observed in shake flask cultures. At a higher agitation speed (600 rpm), the highest curdlan production (64.4 g/l) was obtained in 120 h of a batch fermentation. However, curdlan production was not improved at the higher agitation speed (700 rpm). For the mass production of curdlan, fermentation was performed in a 300-liter fermenter under the condition where the same volumetric oxygen transfer coefficient was obtained as in 5-liter jar fermentation. As high as 9.28 kg of curdlan with a final concentration of 58 g/l was obtained in 120 h batch cultivation, enlarging the potential in the industrial production of curdlan.

A Novel Small Molecule Facilitates the Reprogramming of Human Somatic Cells into a Pluripotent State and Supports the Maintenance of an Undifferentiated State of Human Pluripotent Stem Cells

Booster of pluripotency: RSC133, a new synthetic derivative of indoleacrylic acid/indolepropionic acid, exhibits dual activity by inhibiting histone deacetylase and DNA methyltransferase. Furthermore it potently improves the reprogramming of human somatic cells into a pluripotent state and aids the growth and maintenance of human pluripotent stem cells (hPSCs).

Synthesis of mini-proinsulin precursors using N-termini of human TNF α as fusion partners in recombinant Escherichia coli

Synthesis of human mini-proinsulin precursors was investigated in controlled fed-batch cultures at high cell concentrations of recombinant Escherichia coli. Transcription of the recombinant gene was controlled by a T7 promoter system. The human mini-proinsulin was prepared by substituting a C-chain peptide of natural proinsulin with a peptide sequence of only nine amino acids. The reduced size of fusion proinsulin and hence the increased purity of human insulin in the recombinant product may contribute to increasing the fermentation yield of human insulin. Three precursors (T1-, T2-, and T3-M2PI) were constructed by utilizing the N-terminus residues of human tumor necrosis factor α as fusion partners. The T2 precursor was most soluble in the cytoplasm, and exerted the most inhibitory effect on recombinant cell growth. In the production of T2-M2PI, significant amounts of undesirable metabolic by-products (acetate and ammonia) accumulated in the culture broth even at very low specific cell growth rate. The major portion of all synthesized precursors aggregated to insoluble inclusion bodies but the protein aggregates were easily converted to monomers in the presence of the anionic detergent (SDS) without using any reducing agent. With the expression of T1-M2PI, growth inhibition was minimal, and the maximum volumetric yield of mini-proinsulin (M2PI) in fermentation cultures was at the highest level among the synthesized precursors.

Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair

Summary Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system.

Factors affecting in vivo viability of DNA-injected bovine blastocysts produced in vitro

In vitro matured and fertilized bovine ova were microinjected with pBL1, which consisted of the bovine beta-casein gene promoter, human lactoferrin cDNA and SV40 polyadenylation signal. Of the 2931 zygotes injected, 2505 (85.5%) survived 1 h after DNA injection and were cultured in 50-microl drops of CR1aa medium containing 3 mg/ml BSA under mineral oil at 39 degrees C, 5% CO2 in air. Cleaved (2- to 8-cell) embryos were selected at approximately 48 h after DNA injection and then cultured further in 50-microl drops of CR1aa medium supplemented with 10% (v/v) FBS. Blastocysts were classified into 4 quality grades and 3 developmental stages by morphological criteria. Then all but poor quality blastocysts were nonsurgically transferred to the uterus of heifers 7 to 8 d after natural estrus. Following transfer, the recipients were observed for signs of estrus, and pregnancy was confirmed by palpation per rectum at approximately 60 d of gestation. Although 72.0% (1804/2505 ) of the DNA-injected zygotes reached 2- to 8-cell stages only 5.2% (131/2505) developed to blastocysts. A total of 75 DNA-injected, in vitro cultured blastocysts were transferred to 59 recipients. When 2 blastocysts were transferred to a single recipient, only the better quality embryo was counted. The overall pregnancy rate was 30.5% (18/59 ) and reflected 1) an apparent correlation between the quality of embryos and the pregnancy rate. However, the difference was not statistically significant. 2) expanded blastocysts had a higher pregnancy rate (50.0%, 11/22 ) than early (13.3%, 2 15 ) or mid (22.7%, 5/22 ) blastocysts with a significant difference between expanded and early blastocysts (P < 0.05). 3) the pregnancy rate of DNA-injected blastocysts was higher when they were transferred at Day 7 (34.5%, 10/29 ) or 8 (36.8%, 7/19 ) than at Day 6 (9.0%, 1/11 ). The results indicate that the developmental stage of DNA-injected bovine embryos may be one of contributing factors in improving the pregnancy rate after transfer, although the effects of the quality and culture period of the embryos may not be inconsequential.

The unique spliceosome signature of human pluripotent stem cells is mediated by SNRPA1, SNRPD1, and PNN

Spliceosomes are the core host of pre-mRNA splicing, allowing multiple protein isoforms to be produced from a single gene. Herein, we reveal that spliceosomes are more abundant in human pluripotent stem cells (hPSs), including human embryonic stem cells (hESs) and human induced pluripotent stem cells (hiPSs), than non-hPSs, and their presence is associated with high transcriptional activity. Supportively, spliceosomal components involved in the catalytically active pre-mRNA splicing step were mainly co-localized with hPS spliceosomes. By profiling the gene expression of 342 selected splicing factors, we found that 71 genes were significantly altered during the reprogramming of human somatic cells into hiPSs. Among them, SNRPA1, SNRPD1, and PNN were significantly up-regulated during the early stage of reprogramming, identified as hub genes by interaction network and cluster analysis. SNRPA1, SNRPD1, or PNN depletion led to a pronounced loss of pluripotency and significantly blocked hiPS generation. SNRPA1, SNRPD1, and PNN co-localized with the hPS spliceosomes, physically interacted with each other, and positively influenced the appearance of hPS spliceosomes. Our data suggest that SNRPA1, SNRPD1, and PNN are key players in the regulation of pluripotency-specific spliceosome assembly and the acquisition and maintenance of pluripotency.

DSG2 Is a Functional Cell Surface Marker for Identification and Isolation of Human Pluripotent Stem Cells

Summary Pluripotent stem cells (PSCs) represent the most promising clinical source for regenerative medicine. However, given the cellular heterogeneity within cultivation and safety concerns, the development of specific and efficient tools to isolate a pure population and eliminate all residual undifferentiated PSCs from differentiated derivatives is a prerequisite for clinical applications. In this study, we raised a monoclonal antibody and identified its target antigen as desmoglein-2 (DSG2). DSG2 co-localized with human PSC (hPSC)-specific cell surface markers, and its expression was rapidly downregulated upon differentiation. The depletion of DSG2 markedly decreased hPSC proliferation and pluripotency marker expression. In addition, DSG2-negative population in hPSCs exhibited a notable suppression in embryonic body and teratoma formation. The actions of DSG2 in regulating the self-renewal and pluripotency of hPSCs were predominantly exerted through the regulation of β-catenin/Slug-mediated epithelial-to-mesenchymal transition. Our results demonstrate that DSG2 is a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal.

ESRP1‐Induced CD44 v3 Is Important for Controlling Pluripotency in Human Pluripotent Stem Cells

The importance of alternative splicing (AS) events in pluripotency regulation has been highlighted by the determination of different roles and contributions of different splice isoforms of pluripotency‐related genes and by the identification of distinct pluripotency‐related splicing factors. In particular, epithelial splicing regulatory protein 1 (ESRP1) has been characterized as an essential splicing factor required for the regulation of human pluripotency and differentiation. Nevertheless, a detailed molecular characterization of ESRP1 (mRNA splice variants 1–6) in human pluripotency is lacking. In this study, we determined that ESRP1 splice variants are differentially expressed in undifferentiated and differentiated human pluripotent stem cells (PSCs). Undifferentiated human PSCs predominantly expressed the ESRP1 v1, v4, and v5, and their expression was downregulated upon differentiation. Ectopic expression of ESRP1 v1, v4, or v5 enhanced the pluripotent reprogramming of human fibroblasts and restored the ESRP1 knockdown‐mediated reduction of reprogramming efficiency. Notably, undifferentiated human PSCs expressed the cell surface protein CD44 variant 3 (CD44 v3), and isoform switching from CD44 v3 to CD44 variant 6 (CD44 v6) occurred upon differentiation. Importantly, the human PSC‐specific ESRP1 variants influenced CD44 v3 expression. CD44 knockdown or inhibition of binding of CD44 with its major ligand, hyaluronan, significantly induced the loss of human PSC pluripotency and the reduction of reprogramming efficiency. Our results demonstrate that the effect of ESRP1 and CD44 on human PSC pluripotency is isoform‐dependent and that ESRP1‐induced CD44 v3 is functionally associated with human PSC pluripotency control. Stem Cells 2018;36:1525–1534

Pharmacological Regulation of Oxidative Stress in Stem Cells

Oxidative stress results from an imbalance between reactive oxygen species (ROS) production and antioxidant defense mechanisms. The regulation of stem cell self-renewal and differentiation is crucial for early development and tissue homeostasis. Recent reports have suggested that the balance between self-renewal and differentiation is regulated by the cellular oxidation-reduction (redox) state; therefore, the study of ROS regulation in regenerative medicine has emerged to develop protocols for regulating appropriate stem cell differentiation and maintenance for clinical applications. In this review, we introduce the defined roles of oxidative stress in pluripotent stem cells (PSCs) and hematopoietic stem cells (HSCs) and discuss the potential applications of pharmacological approaches for regulating oxidative stress in regenerative medicine.