Generation of integration-free human induced pluripotent stem cells from a patient with sporadic Parkinson's disease.

A human induced pluripotent stem cell (iPSC) line (KKUi002-A) was generated from a skin fibroblast of a 57-years-old (at sampling) male patient diagnosed with a sporadic Parkinson's disease (PD). A non-integration system was used to reprogram fibroblasts into iPSCs by an episomal vector (OCT4/p53, SOX2/KLF4, L-MYC/LIN28). The KKUi002-A iPSCs displayed typical iPSC morphology, expressed pluripotency markers, differentiated into derivatives of three germ layers, and had a normal karyotype. These PD-derived iPSCs can be used to understand the mechanism underlying PD pathogenesis.

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.

Inhibition of Class I Histone Deacetylase Enhances Self-Reprogramming of Spermatogonial Stem Cells into Pluripotent Stem Cells.

Background and Objectives: Spermatogonial stem cells (SSCs) are the most primitive cells in spermatogenesis and are the only adult stem cells capable of passing on the genome of a given species to the next generation. SSCs are the only adult stem cells known to exhibit high Oct4 expression and can be induced to self-reprogram into pluripotent cells depending on culture conditions. Epigenetic modulation is well known to be involved in the induction of pluripotency of somatic cells. However, epigenetic modulation in self-reprogramming of SSCs into pluripotent cells has not been studied. Methods and Results: In this study, we examined the involvement of epigenetic modulation by assessing whether self-reprogramming of SSCs is enhanced by treatment with epigenetic modulators. We found that second-generation selective class I HDAC inhibitors increased SSC reprogramming efficiency, whereas non-selective HDAC inhibitors had no effect. Conclusions: We showed that pluripotent stem cells derived from adult SSCs by treatment with small molecules with epigenetic modulator functions exhibit pluripotency in vitro and in vivo. Our results suggest that the mechanism of SSC reprogramming by epigenetic modulator can be used for important applications in epigenetic reprogramming research.

The role of microRNA-325-3p as a critical player in cell death in NSCs and astrocytes.

Neural stem cells (NSCs) are defined by their ability to self-renew and generate various cell types within the nervous system. Understanding the underlying mechanism by which NSCs proliferate and differentiate is crucial for the efficient modulation of in vivo neurogenesis. MicroRNAs are small non-coding RNAs controlling gene expression concerned in post-transcriptional control by blocking messenger RNA (mRNA) translation or degrading mRNA. MicroRNAs play a role as modulators by matching target mRNAs. Recent studies have discussed the biological mechanism of microRNA regulation in neurogenesis. To investigate the role of microRNAs in NSCs and NSC-derived glial cells, we screened out NSC-specific microRNAs by using miRNome-wide screening. Then, we induced downregulation by the sponge against the specific microRNA to evaluate the functional role of the microRNA in proliferation, differentiation, and apoptosis in NSCs and NSC-derived astrocytes. We found that microRNA-325-3p is highly expressed in NSCs and astrocytes. Furthermore, we showed that microRNA-325-3p is a regulator of apoptosis by targeting brain-specific angiogenesis inhibitor (BAI1), which is a receptor for apoptotic cells and expressed in the brain and cultured astrocytes. Downregulation of microRNA-325-3p using an inducible sponge system induced cell death by regulating BAI1 in NSCs and NSC-derived astrocytes. Overall, our findings can provide an insight into the potential roles of NSC-specific microRNAs in brain neurogenesis and suggest the possible usage of the microRNAs as biomarkers of neurodegenerative disease.

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.

Effect of an Endoplasmic Reticulum Retention Signal Tagged to Human Anti-Rabies mAb SO57 on Its Expression in Arabidopsis and Plant Growth.

Transgenic Arabidopsis thaliana expressing an anti-rabies monoclonal antibody (mAb), SO57, was obtained using Agrobacterium-mediated floral dip transformation. The endoplasmic reticulum (ER) retention signal Lys-Asp-Glu-Leu (KDEL) was tagged to the C-terminus of the anti-rabies mAb heavy chain to localize the mAb to the ER and enhance its accumulation. When the inaccurately folded proteins accumulated in the ER exceed its storage capacity, it results in stress that can affect plant development and growth. We generated T1 transformants and obtained homozygous T3 seeds from transgenic Arabidopsis to investigate the effect of KDEL on plant growth. The germination rate did not significantly differ between plants expressing mAb SO57 without KDEL (SO plant) and mAb SO57 with KDEL (SOK plant). The primary roots of SOK agar media grown plants were slightly shorter than those of SO plants. Transcriptomic analysis showed that expression of all 11 ER stress-related genes were not significantly changed in SOK plants relative to SO plants. SOK plants showed approximately three-fold higher mAb expression levels than those of SO plants. Consequently, the purified mAb amount per unit of SOK plant biomass was approximately three times higher than that of SO plants. A neutralization assay revealed that both plants exhibited efficient rapid fluorescent focus inhibition test values against the rabies virus relative to commercially available human rabies immunoglobulins. KDEL did not upregulate ER stress-related genes; therefore, the enhanced production of the mAb did not affect plant growth. Thus, KDEL fusion is recommended for enhancing mAb production in plant systems.

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 OCT4-EGFP, NANOG-tdTomato dual reporter human induced pluripotent stem cell line, KKUi001-A, using the CRISPR/Cas9 system.

OCT4 and NANOG are core transcription factor genes in self-renewal, differentiation, and reprogramming. Here, we generated an OCT4-EGFP, NANOG-tdTomato dual reporter hiPSC line, KKUi001-A, on the basis of human induced pluripotent stem cells using CRISPR/Cas9 technology. EGFP and tdTomato reporter were inserted into before the stop codon of OCT4 and NANOG, respectively. Simultaneous expression of EGFP and tdTomato was observed when expression of OCT4 and NANOG was changed during differentiation and reprogramming. KKUi001-A hiPSC line will be a useful tool to find initial time point of OCT4 and NANOG expression during reprogramming process and to screen small molecules that promote reprogramming.

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 spermatogonial stem cells into pluripotent stem cells in the spheroidal state.

Spermatogonial stem cells (SSCs) are unipotent adult stem cells, capable of differentiating into sperm cells. SSCs can be cultured in vitro for a long time. SSCs expressing Oct4, a pluripotency marker, and are the only adult cells which pluripotency can be induced under defined culture conditions. However, because 2D culture imposes limitations in cell junction formation, cell shape, metabolism, response to stimuli, and cell interface with medium, mechanistic studies on reprogramming of SSCs using feeder cells still have many challenges. Recent studies have shown that a culture system using a bio-matrix can be used in long-term feeder-free SSCs culture and for induction of pluripotency in SSCs. However, the bio-matrix cannot be the optimal micro-environment in mechanistic studies because it creates a physical barrier to growth factors and other signaling molecules. To overcome this effect of the matrix, we reprogrammed SSCs into pluripotent ESC-like cells, so-called germline-derived pluripotent stem cells (gPSCs) by using a 3D scaffold, in which cells are less responsive to external stimuli than in 2D cultures. Thus, we confirm the possibility of SSC reprogramming in the spheroidal state and suggest the utility of 3D scaffolds as a tool for studying the mechanism of SSC reprogramming into gPSCs without a bio-matrix.

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.

Metastable Reprogramming State of Single Transcription Factor-Derived Induced Hepatocyte-Like Cells.

We previously described the generation of induced hepatocyte-like cells (iHeps) using the hepatic transcription factor Hnf1a together with small molecules. These iHeps represent a hepatic state that is more mature compared with iHeps generated with multiple hepatic factors. However, the underlying mechanism of hepatic conversion involving transgene dependence of the established iHeps is largely unknown. Here, we describe the generation of transgene-independent iHeps by inducing the ectopic expression of Hnf1a using both an episomal vector and a doxycycline-inducible lentivirus. In contrast to iHeps with sustained expression of Hnf1a, transgene-independent Hnf1a iHeps lose their typical morphology and in vitro functionality with rapid downregulation of hepatic markers upon withdrawal of small molecules. Taken together, our data indicates that the reprogramming state of single factor Hnf1a-derived iHeps is metastable and that the hepatic identity of these cells could be maintained only by the continuous supply of either small molecules or the master hepatic factor Hnf1a. Our findings emphasize the importance of a factor screening strategy for inducing specific cellular identities with a stable reprogramming state in order to eventually translate direct conversion technology to the clinic.

Alteration of Genomic Imprinting Status of Human Parthenogenetic Induced Pluripotent Stem Cells during Neural Lineage Differentiation.

BACKGROUND AND OBJECTIVES: Genomic imprinting modulates growth and development in mammals and is associated with genetic disorders. Although uniparental embryonic stem cells have been used to study genomic imprinting, there is an ethical issue associated with the destruction of human embryos. In this study, to investigate the genomic imprinting status in human neurodevelopment, we used human uniparental induced pluripotent stem cells (iPSCs) that possessed only maternal alleles and differentiated into neural cell lineages. METHODS: Human somatic iPSCs (hSiPSCs) and human parthenogenetic iPSCs (hPgiPSCs) were differentiated into neural stem cells (NSCs) and named hSi-NSCs and hPgi-NSCs respectively. DNA methylation and gene expression of imprinted genes related neurodevelopment was analyzed during reprogramming and neural lineage differentiation. RESULTS: The DNA methylation and expression of imprinted genes were altered or maintained after differentiation into NSCs. The imprinting status in NSCs were maintained after terminal differentiation into neurons and astrocytes. In contrast, gene expression was differentially presented in a cell type-specific manner. CONCLUSIONS: This study suggests that genomic imprinting should be determined in each neural cell type because the genomic imprinting status can differ in a cell type-specific manner. In addition, the in vitro model established in this study would be useful for verifying the epigenetic alteration of imprinted genes which can be differentially changed during neurodevelopment in human and for screening novel imprinted genes related to neurodevelopment. Moreover, the confirmed genomic imprinting status could be used to find out an abnormal genomic imprinting status of imprinted genes related with neurogenetic disorders according to uniparental genotypes.

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.

Two-Step Generation of Oligodendrocyte Progenitor Cells From Mouse Fibroblasts for Spinal Cord Injury.

Oligodendrocyte progenitor cells (OPCs) are attracting attention as the ideal cell therapy for spinal cord injury (SCI). Recently, advanced reprogramming and differentiation techniques have made it possible to generate therapeutic cells for treating SCI. In the present study, we used directly-induced neural stem cells (DNSCs) from fibroblasts to establish OPCs (DN-OPCs) capable of proliferation and confirmed their OPC-specific characteristics. Also, we evaluated the effect of transplanted DN-OPCs on SCI in rats. The DN-OPCs exhibited an OPC-specific phenotype and electrophysiological function and could be differentiated into oligodendrocytes. In the SCI model, transplanted DN-OPCs improved behavior recovery, and showed engraftment into the host spinal cord with expression of myelin basic protein. These results suggest that DN-OPCs could be a new source of potentially useful cells for treating SCI.