Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

KEY MESSAGE: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

Homogeneity of XEN Cells Is Critical for Generation of Chemically Induced Pluripotent Stem Cells.

In induced pluripotent stem cells (iPSCs), pluripotency is induced artificially by introducing the transcription factors Oct4, Sox2, Klf4, and c-Myc. When a transgene is introduced using a viral vector, the transgene may be integrated into the host genome and cause a mutation and cancer. No integration occurs when an episomal vector is used, but this method has a limitation in that remnants of the virus or vector remain in the cell, which limits the use of such iPSCs in therapeutic applications. Chemical reprogramming, which relies on treatment with small-molecule compounds to induce pluripotency, can overcome this problem. In this method, reprogramming is induced according to the gene expression pattern of extra-embryonic endoderm (XEN) cells, which are used as an intermediate stage in pluripotency induction. Therefore, iPSCs can be induced only from established XEN cells. We induced XEN cells using small molecules that modulate a signaling pathway and affect epigenetic modifications, and devised a culture method in which can be produced homogeneous XEN cells. At least 4 passages were required to establish morphologically homogeneous chemically induced XEN (CiXEN) cells, whose properties were similar to those of XEN cells, as revealed through cellular and molecular characterization. Chemically iPSCs derived from CiXEN cells showed characteristics similar to those of mouse embryonic stem cells. Our results show that the homogeneity of CiXEN cells is critical for the efficient induction of pluripotency by chemicals.

Enhancement of proliferation of human umbilical cord blood-derived CD34+ hematopoietic stem cells by a combination of hyper-interleukin-6 and small molecules.

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB-HSCs) in transplantation, however, is the low numbers of HSCs in a unit of cord blood. To overcome this limitation, various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study, we investigated a synergistic effect of the combination of HIL-6, SR1, and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34+ cells. These results suggest that the combination of SR1, UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus, SR1, UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation.

Plant-derived human recombinant growth factors and serum albumin maintain stemness of human-induced pluripotent stem cells.

Stem cells are an important therapeutic source for recovery and regeneration, as their ability of self-renewal and differentiation offers an unlimited supply of highly specialized cells for therapeutic transplantation. Growth factors and serum are essential for maintaining the characteristics of stem cells in culture and for inducing differentiation. Because growth factors are produced mainly in bacterial (Escherichia coli) or animal cells, the use of such growth factors raises safety concerns that need to be resolved for the commercialization of stem cell therapeutics. To overcome this problem, studies on proteins produced in plants have been conducted. Here, we describe the functions of plant-derived fibroblast growth factor 2 (FGF2) and human serum albumin in the maintenance and differentiation of human-induced pluripotent stem cells (hiPSCs). Plant-derived FGF2 and human epidermal growth factor EGF were able to differentiate hiPSCs into neural stem cells (NSCs). These NSCs could differentiate into neuronal and glial cells. Our results imply that culturing stem cells in animal-free culture medium, which is composed of plant-derived proteins, would facilitate stem cell application research, for example, for cell therapy, by reducing contamination risk.

Establishment of an integration-free human induced pluripotent stem cell line (TJCi001-A) from normal bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) possess excellent therapeutic potential for the treatment of various diseases including graft-versus-host disease, rheumatoid arthritis, osteoarthritis, and multiple sclerosis. Here, we generated an induced pluripotent stem cell (iPSC) line from BM-MSCs employing a non-integrating episomal vector. The generated iPSCs expressed pluripotency markers, showed a normal karyotype, and exhibited the potential for in vitro differentiation into three germ layers. This iPSC line can be used as a healthy control in stem cell therapeutics and disease modeling studies.

Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant.

Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1-10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration.

Coadministration of endothelial and smooth muscle cells derived from human induced pluripotent stem cells as a therapy for critical limb ischemia.

Critical limb ischemia is a condition in which tissue necrosis occurs due to arterial occlusion, resulting in limb amputation in severe cases. Both endothelial cells (ECs) and vascular smooth muscle cells (SMCs) are needed for the regeneration of peripheral arteries in ischemic tissues. However, it is difficult to isolate and cultivate primary EC and SMC from patients for therapeutic angiogenesis. Induced pluripotent stem cells (iPSCs) are regarded as useful stem cells due to their pluripotent differentiation potential. In this study, we explored the therapeutic efficacy of human iPSC-derived EC and iPSC-derived SMC in peripheral artery disease model. After the induction of mesodermal differentiation of iPSC, CD34+ progenitor cells were isolated by magnetic-activated cell sorting. Cultivation of the CD34+ progenitor cells in endothelial culture medium induced the expression of endothelial markers and phenotypes. Moreover, the CD34+ cells could be differentiated into SMC by cultivation in SMC culture medium. In a murine hindlimb ischemia model, cotransplantation of EC with SMC improved blood perfusion and increased the limb salvage rate in ischemic limbs compared to transplantation of either EC or SMC alone. Moreover, cotransplantation of EC and SMC stimulated angiogenesis and led to the formation of capillaries and arteries/arterioles in vivo. Conditioned medium derived from SMC stimulated the migration, proliferation, and tubulation of EC in vitro, and these effects were recapitulated by exosomes isolated from the SMC-conditioned medium. Together, these results suggest that iPSC-derived SMC enhance the therapeutic efficacy of iPSC-derived EC in peripheral artery disease via an exosome-mediated paracrine mechanism.

A Plant-Derived Antigen-Antibody Complex Induces Anti-Cancer Immune Responses by Forming a Large Quaternary Structure.

The antigen-antibody complex (AAC) has novel functions for immunomodulation, encouraging the application of diverse quaternary protein structures for vaccination. In this study, GA733 antigen and anti-GA733 antibody proteins were both co-expressed to obtain the AAC protein structures in a F1 plant obtained by crossing the plants expressing each protein. In F1 plant, the antigen and antibody assembled to form a large quaternary circular ACC structure (~30 nm). The large quaternary protein structures induced immune response to produce anticancer immunoglobulins G (IgGs) that are specific to the corresponding antigens in mouse. The serum containing the anticancer IgGs inhibited the human colorectal cancer cell growth in the xenograft nude mouse. Taken together, antigens and antibodies can be assembled to form AAC protein structures in plants. Plant crossing represents an alternative strategy for the formation of AAC vaccines that efficiently increases anticancer antibody production.

Generation of human androgenetic induced pluripotent stem cells.

In humans, parthenogenesis and androgenesis occur naturally in mature cystic ovarian teratomas and androgenetic complete hydatidiform moles (CHM), respectively. Our previous study has reported human parthenogenetic induced pluripotent stem cells from ovarian teratoma-derived fibroblasts and screening of imprinted genes using genome-wide DNA methylation analysis. However, due to the lack of the counterparts of uniparental cells, identification of new imprinted differentially methylated regions has been limited. CHM are inherited from only the paternal genome. In this study, we generated human androgenetic induced pluripotent stem cells (AgHiPSCs) from primary androgenetic fibroblasts derived from CHM. To investigate the pluripotency state of AgHiPSCs, we analyzed their cellular and molecular characteristics. We tested the DNA methylation status of imprinted genes using bisulfite sequencing and demonstrated the androgenetic identity of AgHiPSCs. AgHiPSCs might be an attractive alternative source of human androgenetic embryonic stem cells. Furthermore, AgHiPSCs can be used in regenerative medicine, for analysis of genomic imprinting, to study imprinting-related development, and for disease modeling in humans.

Reprogramming of Cancer Cells into Induced Pluripotent Stem Cells Questioned.

BACKGROUND AND OBJECTIVES: Several recent studies have claimed that cancer cells can be reprogrammed into induced pluripotent stem cells (iPSCs). However, in most cases, cancer cells seem to be resistant to cellular reprogramming. Furthermore, the underlying mechanisms of limited reprogramming in cancer cells are largely unknown. Here, we identified the candidate barrier genes and their target genes at the early stage of reprogramming for investigating cancer reprogramming. METHODS: We tried induction of pluripotency in normal human fibroblasts (BJ) and both human benign (MCF10A) and malignant (MCF7) breast cancer cell lines using a classical retroviral reprogramming method. We conducted RNA-sequencing analysis to compare the transcriptome of the three cell lines at early stage of reprogramming. RESULTS: We could generate iPSCs from BJ, whereas we were unable to obtain iPSCs from cancer cell lines. To address the underlying mechanism of limited reprogramming in cancer cells, we identified 29 the candidate barrier genes based on RNA-sequencing data. In addition, we found 40 their target genes using Cytoscape software. CONCLUSIONS: Our data suggest that these genes might one of the roadblock for cancer cell reprogramming. Furthermore, we provide new insights into application of iPSCs technology in cancer cell field for therapeutic purposes.

Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts.

Generation of induced pluripotent stem cells (iPSCs) by defined factors (OCT4, SOX2, C-MYC, and KLF4) from various human primary cells has been reported. Human fibroblasts have been widely used as a cellular source in reprogramming studies over recent decades. The original method of iPSC generation uses retro- or lentivirus vectors that require integration of viral DNA into the target cells. The integration of exogenous genes encoding transcription factors (OCT4, SOX2, C-MYC, and KLF4) can be detected in iPSCs, raising concern about the risk of mutagenesis and tumor formation. Therefore, stem cell therapy would ideally require generation of integration-free iPSCs using non-integration gene delivery system such as Sendai virus, recombinant proteins, synthetic mRNA, and episomal vectors. Several groups have reported that episomal vectors are capable of reprogramming human fibroblasts into iPSCs. Although vector concentration and cell density are important in the episomal vector reprogramming method, optimization of this method for human fibroblasts has not been reported. In this study, we determined optimal conditions for generating integration-free iPSCs from human fibroblasts through the use of different concentrations of episomal vectors (OCT4/p53, SOX2/KLF4, L-MYC/LIN28A) and different plating cell density. We found that optimized vector concentration and cell density accelerate reprogramming and improve iPSC generation. Our study provides a detailed stepwise protocol for improved generation of integration-free iPSCs from human fibroblasts by transfection with episomal vectors.

Smoking Exposure in Early Pregnancy and Adverse Pregnancy Outcomes: Usefulness of Urinary Tobacco-Specific Nitrosamine Metabolite 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol Levels.

OBJECTIVES: The aim was to investigate the effect of -maternal smoking exposure assessed by urinary tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-a1-butanol (NNAL) with adverse pregnancy outcomes. METHODS: A total of 251 pregnant women were recruited. Urinary cotinine and NNAL were measured. Participants' sociodemographics were obtained by questionnaire and pregnancy outcomes were collected by charts review after delivery. RESULTS: The prevalence of smoking was 8.4% (21 of 249), 1.2% (3 of 241), and 3.7% (9 of 241) in pregnant women according to questionnaire, cotinine, and NNAL, respectively. As compared with questionnaire positivity and cotinine levels, women with positive NNAL were independent determinants for spontaneous abortion (adjusted OR 12.357, 95% CI 2.053-74.368), preterm birth (adjusted OR 22.239, 95% CI 3.737-132.357), and small for gestational age (adjusted OR 6.915, 95% CI 1.385-34.524). CONCLUSIONS: Urinary NNAL might be a useful biomarker in detection of maternal smoking status in association with adverse pregnancy outcomes. Use of this marker in preconception and pregnancy counselling before planning pregnancy may allow prevention of several adverse pregnancy outcomes.

Novel imprinted single CpG sites found by global DNA methylation analysis in human parthenogenetic induced pluripotent stem cells.

Genomic imprinting is the process of epigenetic modification whereby genes are expressed in a parent-of-origin dependent manner; it plays an important role in normal growth and development. Parthenogenetic embryos contain only the maternal genome. Parthenogenetic embryonic stem cells could be useful for studying imprinted genes. In humans, mature cystic ovarian teratomas originate from parthenogenetic activation of oocytes; they are composed of highly differentiated mature tissues containing all three germ layers. To establish human parthenogenetic induced pluripotent stem cell lines (PgHiPSCs), we generated parthenogenetic fibroblasts from ovarian teratoma tissues. We compared global DNA methylation status of PgHiPSCs with that of biparental human induced pluripotent stem cells by using Illumina Infinium HumanMethylation450 BeadChip array. This analysis identified novel single imprinted CpG sites. We further tested DNA methylation patterns of two of these sites using bisulfite sequencing and described novel candidate imprinted CpG sites. These results confirm that PgHiPSCs are a powerful tool for identifying imprinted genes and investigating their roles in human development and diseases.

Multigenerational effects of maternal cigarette smoke exposure during pregnancy on sperm counts of F1 and F2 male offspring.

Animal models and human studies showed that in utero cigarette smoke exposure decreases sperm counts of offspring. This study used a mouse model to investigate the effects of maternal exposure to cigarette smoke on reproductive systems in F1 and F2 male offspring. Female ICR mice were exposed either to clean air or to cigarette smoke during pregnancy at the post-implantation stage. Epididymal sperm counts were decreased in a cigarette smoke dose-dependent manner in F1 (by 40-60%) and F2 males (by 23-40%) at postnatal day 56. In F1, the seminiferous epithelium heights were lower in the cigarette smoke-exposed groups than in the control group, and these effects were sustained in F2 males. Results suggest that maternal cigarette smoke exposure during pregnancy can have a multigenerational adverse effect on sperm counts in male offspring, which is mediated through in utero exposure of fetal germ cells to cigarette smoke.

DJ-1 Regulates Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle-like Cells in Response to Sphingosylphosphorylcholine.

Although multiple factors contribute to the differentiation of human mesenchymal stem cells (hMSCs) into various types of cells, the differentiation of hMSCs into smooth muscle cells (SMCs), one of central events in vascular remodeling, remains to be clarified. ROS participate in the differentiation of hMSCs into several cell types and were regulated by redox-sensitive molecules including a multifunctional protein DJ-1. Here, we investigated the correlation between altered proteins, especially those related to ROS, and SMC differentiation in sphingosylphosphorylcholine (SPC)-stimulated hMSCs. Treatment with SPC resulted in an increased expression of SMC markers, namely α-smooth muscle actin (SMA) and calponin, and an increased production of ROS in hMSCs. A proteomic analysis of SPC-stimulated hMSCs revealed a distinctive alteration of the ratio between the oxidized and reduced forms of DJ-1 in hMSCs in response to SPC. The increased abundance of oxidized DJ-1 in SPC-stimulated hMSCs was validated by immunoblot analysis. The SPC-induced increase in the expression of α-SMA was stronger in DJ-1-knockdown hMSCs than in control cells. Moreover, the expression of α-SMA, and the calponin and generation of ROS in response to SPC were weaker in normal hMSCs than in DJ-1-overexpressing hMSCs. Exogenous H2 O2 mimicked the responses induced by SPC treatment. These results indicate that the ROS-related DJ-1 pathway regulates the differentiation of hMSCs into SMCs in response to SPC.

Epigenetic alteration of imprinted genes during neural differentiation of germline-derived pluripotent stem cells.

Spermatogonial stem cells (SSCs), which are unipotent stem cells in the testes that give rise to sperm, can be converted into germline-derived pluripotent stem (gPS) by self-induction. The androgenetic imprinting pattern of SSCs is maintained even after their reprogramming into gPS cells. In this study, we used an in vitro neural differentiation model to investigate whether the imprinting patterns are maintained or altered during differentiation. The androgenetic patterns of H19, Snrpn, and Mest were maintained even after differentiation of gPS cells into NSCs (gPS-NSCs), whereas the fully unmethylated status of Ndn in SSCs was altered to somatic patterns in gPS cells and gPS-NSCs. Thus, our study demonstrates epigenetic alteration of genomic imprinting during the induction of pluripotency in SSCs and neural differentiation, suggesting that gPS-NSCs can be a useful model to study the roles of imprinted genes in brain development and human neurodevelopmental disorders.

Ectopic overexpression of Nanog induces tumorigenesis in non-tumorous fibroblasts.

Key regulatory genes in pluripotent stem cells are of interest not only as reprogramming factors but also as regulators driving tumorigenesis. Nanog is a transcription factor involved in the maintenance of embryonic stem cells and is one of the reprogramming factors along with Oct4, Sox2, and Lin28. Nanog expression has been detected in different types of tumors, and its expression is a poor prognosis for cancer patients. However, there is no clear evidence that Nanog is functionally involved in tumorigenesis. In this study, we induced overexpression of Nanog in mouse embryonic fibroblast cells and subsequently assessed their morphological changes, proliferation rate, and tumor formation ability. We found that Nanog overexpression induced immortalization of mouse embryonic fibroblast cells (MEFs) and increased their proliferation rate in vitro. We also found that formation of tumors after subcutaneous injection of retroviral-Nanog infected MEFs (N-MEFs) into athymic mouse. Cancer-related genes such as Bmi1 were expressed at high levels in N-MEFs. Hence, our results demonstrate that Nanog is able to transform normal somatic cells into tumor cells.

In vitro generation of functional dendritic cells differentiated from CD34 negative cells isolated from human umbilical cord blood.

Dendritic cells (DCs) are the most potent antigen-presenting cells that play a crucial role in the initiation of an immune response. As DC-based therapeutic applications is increasing, large-scale DC production is required for transplantation. Human umbilical cord blood (UCB) has been shown to contain a rare and precious population of hematopoietic stem cells (HSCs), which can give rise to DCs. The CD34 antigen has been widely used as a cell surface marker to identify HSCs. In this study, we used CD34 antibody to isolate CD34(+) and CD34(-) cells and compared the ability to differentiate into DCs. We used a two-step method combined with the magnetic bead sorting system to isolate CD34(+) and CD34(-) cells from human UCB. Analysis of cellular properties and functionality using a migration assay and T cell proliferation assay revealed no significant differences between CD34(+) cells and CD34(-) cells in their ability to generate DCs.

Therapeutic potential of induced neural stem cells for spinal cord injury.

The spinal cord does not spontaneously regenerate, and treatment that ensures functional recovery after spinal cord injury (SCI) is still not available. Recently, fibroblasts have been directly converted into induced neural stem cells (iNSCs) by the forced expression defined transcription factors. Although directly converted iNSCs have been considered to be a cell source for clinical applications, their therapeutic potential has not yet been investigated. Here we show that iNSCs directly converted from mouse fibroblasts enhance the functional recovery of SCI animals. Engrafted iNSCs could differentiate into all neuronal lineages, including different subtypes of mature neurons. Furthermore, iNSC-derived neurons could form synapses with host neurons, thus enhancing the locomotor function recovery. A time course analysis of iNSC-treated SCI animals revealed that engrafted iNSCs effectively reduced the inflammatory response and apoptosis in the injured area. iNSC transplantation also promoted the active regeneration of the endogenous recipient environment in the absence of tumor formation. Therefore, our data suggest that directly converted iNSCs hold therapeutic potential for treatment of SCI and may thus represent a promising cell source for transplantation therapy in patients with SCI.

A novel feeder-free culture system for expansion of mouse spermatogonial stem cells.

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.