CDISC-compliant clinical trial imaging management system with automatic verification and data Transformation: Focusing on tumor response assessment data in clinical trials.

OBJECTIVE: Major issues in imaging data management of tumor response assessment in clinical trials include high human errors in data input and unstandardized data structures, warranting a new breakthrough IT solution. Thus, we aim to develop a Clinical Data Interchange Standards Consortium (CDISC)-compliant clinical trial imaging management system (CTIMS) with automatic verification and transformation modules for implementing the CDISC Study Data Tabulation Model (SDTM) in the tumor response assessment dataset of clinical trials. MATERIALS AND METHODS: In accordance with various CDISC standards guides and Response Evaluation Criteria in Solid Tumors (RECIST) guidelines, the overall system architecture of CDISC-compliant CTIMS was designed. Modules for standard-compliant electronic case report form (eCRF) to verify data conformance and transform into SDTM data format were developed by experts in diverse fields such as medical informatics, medical, and clinical trial. External validation of the CDISC-compliant CTIMS was performed by comparing it with our previous CTIMS based on real-world data and CDISC validation rules by Pinnacle 21 Community Software. RESULTS: The architecture of CDISC-compliant CTIMS included the standard-compliant eCRF module of RECIST, the automatic verification module of the input data, and the SDTM transformation module from the eCRF input data to the SDTM datasets based on CDISC Define-XML. This new system was incorporated into our previous CTIMS. External validation demonstrated that all 176 human input errors occurred in the previous CTIMS filtered by a new system yielding zero error and CDISC-compliant dataset. The verified eCRF input data were automatically transformed into the SDTM dataset, which satisfied the CDISC validation rules by Pinnacle 21 Community Software. CONCLUSIONS: To assure data consistency and high quality of the tumor response assessment data, our new CTIMS can minimize human input error by using standard-compliant eCRF with an automatic verification module and automatically transform the datasets into CDISC SDTM format.

A combination of small molecules directly reprograms mouse fibroblasts into neural stem cells.

The generation of induced neural stem cells (iNSCs) from somatic cells using defined factors provides new avenues for basic research and cell therapies for various neurological diseases, such as Parkinson's disease, Huntington's disease, and spinal cord injuries. However, the transcription factors used for direct reprogramming have the potential to cause unexpected genetic modifications, which limits their potential application in cell therapies. Here, we show that a combination of four chemical compounds resulted in cells directly acquiring a NSC identity; we termed these cells chemically-induced NSCs (ciNSCs). ciNSCs expressed NSC markers (Pax6, PLZF, Nestin, Sox2, and Sox1) and resembled NSCs in terms of their morphology, self-renewal, gene expression profile, and electrophysiological function when differentiated into the neuronal lineage. Moreover, ciNSCs could differentiate into several types of mature neurons (dopaminergic, GABAergic, and cholinergic) as well as astrocytes and oligodendrocytes in vitro. Taken together, our results suggest that stably expandable and functional ciNSCs can be directly reprogrammed from mouse fibroblasts using a combination of small molecules without any genetic manipulation, and will provide a new source of cells for cellular replacement therapy of neurodegenerative diseases.

Hypoxic conditioned medium from human amniotic fluid-derived mesenchymal stem cells accelerates skin wound healing through TGF-ß/SMAD2 and PI3K/Akt pathways.

In a previous study, we isolated human amniotic fluid (AF)-derived mesenchymal stem cells (AF-MSCs) and utilized normoxic conditioned medium (AF-MSC-norCM) which has been shown to accelerate cutaneous wound healing. Because hypoxia enhances the wound healing function of mesenchymal stem cell-conditioned medium (MSC-CM), it is interesting to explore the mechanism responsible for the enhancement of wound healing function. In this work, hypoxia not only increased the proliferation of AF-MSCs but also maintained their constitutive characteristics (surface marker expression and differentiation potentials). Notably, more paracrine factors, VEGF and TGF-ß1, were secreted into hypoxic conditioned medium from AF-MSCs (AF-MSC-hypoCM) compared to AF-MSC-norCM. Moreover, AF-MSC-hypoCM enhanced the proliferation and migration of human dermal fibroblasts in vitro, and wound closure in a skin injury model, as compared to AF-MSC-norCM. However, the enhancement of migration of fibroblasts accelerated by AF-MSC-hypoCM was inhibited by SB505124 and LY294002, inhibitors of TGF-ß/SMAD2 and PI3K/AKT, suggesting that AF-MSC-hypoCM-enhanced wound healing is mediated by the activation of TGF-ß/SMAD2 and PI3K/AKT. Therefore, AF-MSC-hypoCM enhances wound healing through the increase of hypoxia-induced paracrine factors via activation of TGF-ß/SMAD2 and PI3K/AKT pathways.

Reprogramming of mouse fibroblasts into induced pluripotent stem cells with Nanog.

Oct4-Sox2-Nanog transcriptional networks are critical for the maintenance of embryonic stem (ES) cell self-renewal and induction of pluripotency. However, in transcription factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), Nanog is initially dispensable and Oct4 remains the sole factor that could not be substituted/omitted. Here, we show that mouse fibroblasts could be reprogrammed into iPSCs by Nanog and Bmi1, which replaces Sox2, Klf4, and c-Myc, in the absence of Oct4. Furthermore, we show that in the presence of shh agonists (oxysterol and purmophamine), which replaces the function of Bmi1, a single transcription factor, Nanog is sufficient to reprogram mouse fibroblasts into iPSCs. Nanog-induced iPSCs resemble mESCs in terms of morphology, global gene expression profiles, epigenetic status and pluripotency both in vitro and in vivo. These findings support that Nanog can replace the Oct4 for the somatic cell reprogramming and underlie the mechanisms of Nanog in reprogramming process.

Human induced neural stem cells support functional recovery in spinal cord injury models.

Spinal cord injury (SCI) is a clinical condition that leads to permanent and/or progressive disabilities of sensory, motor, and autonomic functions. Unfortunately, no medical standard of care for SCI exists to reverse the damage. Here, we assessed the effects of induced neural stem cells (iNSCs) directly converted from human urine cells (UCs) in SCI rat models. We successfully generated iNSCs from human UCs, commercial fibroblasts, and patient-derived fibroblasts. These iNSCs expressed various neural stem cell markers and differentiated into diverse neuronal and glial cell types. When transplanted into injured spinal cords, UC-derived iNSCs survived, engrafted, and expressed neuronal and glial markers. Large numbers of axons extended from grafts over long distances, leading to connections between host and graft neurons at 8 weeks post-transplantation with significant improvement of locomotor function. This study suggests that iNSCs have biomedical applications for disease modeling and constitute an alternative transplantation strategy as a personalized cell source for neural regeneration in several spinal cord diseases.

OCT4-induced oligodendrocyte progenitor cells promote remyelination and ameliorate disease.

The generation of human oligodendrocyte progenitor cells (OPCs) may be therapeutically valuable for human demyelinating diseases such as multiple sclerosis. Here, we report the direct reprogramming of human somatic cells into expandable induced OPCs (iOPCs) using a combination of OCT4 and a small molecule cocktail. This method enables generation of A2B5+ (an early marker for OPCs) iOPCs within 2 weeks retaining the ability to differentiate into MBP-positive mature oligodendrocytes. RNA-seq analysis revealed that the transcriptome of O4+ iOPCs was similar to that of O4+ OPCs and ChIP-seq analysis revealed that putative OCT4-binding regions were detected in the regulatory elements of CNS development-related genes. Notably, engrafted iOPCs remyelinated the brains of adult shiverer mice and experimental autoimmune encephalomyelitis mice with MOG-induced 14 weeks after transplantation. In conclusion, our study may contribute to the development of therapeutic approaches for neurological disorders, as well as facilitate the understanding of the molecular mechanisms underlying glial development.

Nanog-induced dedifferentiation of p53-deficient mouse astrocytes into brain cancer stem-like cells.

Self-renewal, differentiation, and tumorigenicity characterize cancer stem cells (CSCs), which are rare and maintained by specific cell fate regulators. CSCs are isolated from glioblastoma multiforme (GBM) and may be responsible for the lethality of incurable brain tumors. Brain CSCs may arise from the transformation of undifferentiated, nestin-positive neural stem or progenitor cells and GFAP-expressing astrocytes. Here, we report a role of Nanog in the genesis of cancer stem-like cells. Using primary murine p53-knockout astrocytes (p53(-/-) astrocytes), we provide evidence that enforced Nanog expression can increase the cellular growth rate and transform phenotypes in vitro and in vivo. In addition, Nanog drives p53(-/-) astrocytes toward a dedifferentiated, CSC-like phenotype with characteristic neural stem cell/progenitor marker expression, neurosphere formation, self-renewal activity, and tumor development. These findings suggest that Nanog promotes dedifferentiation of p53-deficient mouse astrocytes into cancer stem-like cells by changing the cell fate and transforming cell properties.

Optimal suppression of protein phosphatase 2A activity is critical for maintenance of human embryonic stem cell self-renewal.

The self-renewal of embryonic stem cells involves a balance between processes governed by crosstalk between intrinsic and extrinsic factors. We hypothesized that protein serine/threonine phosphatase 2A (PP2A) may play a central role in the signaling pathways that regulate human embryonic stem cell (hESC) self-renewal. Biochemical analyses revealed that PP2A activity gradually increases over the course of hESC differentiation; PP2A/C and PP2A/A levels also increased. The overexpression of PP2A/C or the addition of PP2A activator C2-ceramide promoted hESC differentiation. Accordingly, the addition of PP2A inactivator okadaic acid (OA) maintained hESC self-renewal in the absence of basic fibroblast growth factor (bFGF). The hESCs maintained with OA expressed pluripotency markers and exhibited substantial telomerase activity with normal karyotypes. The hESCs were able to differentiate into derivatives of the three germ layers, both in vitro and in vivo. Furthermore, the addition of OA and bFGF enabled the maintenance of hESC self-renewal without feeder cells, even in chemically defined xeno-free media. These findings shed a light on the role of PP2A in hESC differentiation and provide a novel strategy for maintaining the self-renewal capability of hESC in bFGF-free, feeder cell-free, and xeno-free media through the optimal suppression of PP2A activity using OA.

Overexpression of Nanog in amniotic fluid-derived mesenchymal stem cells accelerates dermal papilla cell activity and promotes hair follicle regeneration.

Alopecia, one of the most common chronic diseases, can seriously affect a patient's psychosocial life. Dermal papilla (DP) cells serve as essential signaling centers in the regulation of hair growth and regeneration and are associated with crosstalk between autocrine/paracrine factors and the surrounding environment. We previously demonstrated that amniotic fluid-derived mesenchymal stem cell-conditioned medium (AF-MSC-CM) accelerates hair regeneration and growth. The present study describes the effects of overexpression of a reprogramming factor, Nanog, on MSC properties, the paracrine effects on DP cells, and in vivo hair regrowth. First, we examined the in vitro proliferation and lifespan of AF-MSCs overexpressing reprogramming factors, including Oct4, Nanog, and Lin28, alone or in combination. Among these factors, Nanog was identified as a key factor in maintaining the self-renewal capability of AF-MSCs by delaying cellular senescence, increasing the endogenous expression of Oct4 and Sox2, and preserving stemness. Next, we evaluated the paracrine effects of AF-MSCs overexpressing Nanog (AF-N-MSCs) by monitoring secretory molecules related to hair regeneration and growth (IGF, PDGF, bFGF, and Wnt7a) and proliferation of DP cells. In vivo studies revealed that CM derived from AF-N-MSCs (AF-N-CM) accelerated the telogen-to-anagen transition in hair follicles (HFs) and increased HF density. The expression of DP and HF stem cell markers and genes related to hair induction were higher in AF-N-CM than in CM from AF-MSCs (AF-CM). This study suggests that the secretome from autologous MSCs overexpressing Nanog could be an excellent candidate as a powerful anagen inducer and hair growth stimulator for the treatment of alopecia.

Induction of neural stem cell-like cells (NSCLCs) from mouse astrocytes by Bmi1.

Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1.

Induction of serum amyloid A genes is associated with growth and apoptosis of HC11 mammary epithelial cells.

In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFalpha treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IkappaBalpha. Similar results were observed in response to interleukin-1 (IL-1), IL-6 and interferon gamma (IFNgamma). Furthermore, overexpression of the SAA1 and SAA2 isoforms suppressed growth and accelerated apoptosis of HC11 cells by increasing caspase 3/7 and caspase 8 activities, but the apoptotic effect of tumor necrosis factor alpha (TNFalpha) on HC11 cells was not enhanced. We found that expression of SAA1 and SAA2, but not SAA3, was regulated by an NFkappaB-dependent pathway, and that overexpression of SAA isoforms accelerated the apoptosis of HC11 cells.

WDNM1 is associated with differentiation and apoptosis of mammary epithelial cells.

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFalpha treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFalpha induced WDNM1 expression showed the NFkappaB-dependent mechanism since it's expression was abrogated in IkappaBalphaM (super-repressor of NFkappaB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFalpha-NFkappaB signal pathway, is associated with HC11 cell apoptosis.

Additive effect of bFGF and selenium on expansion and paracrine action of human amniotic fluid-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cell-derived conditioned medium (MSC-CM) has emerged as a promising cell-free tool for restoring degenerative diseases and treating traumatic injuries. The present study describes the effect of selenium as a reactive oxygen species (ROS) scavenger and its additive effect with basic fibroblast growth factor (bFGF) on in vitro expansion of amniotic fluid (AF)-MSCs and the paracrine actions of AF-MSC-CM as well as the associated cellular and molecular mechanisms. METHODS: In this study, we obtained CM from human AF-MSCs cultured with selenium. The stemness of selenium-treated AF-MSCs was evaluated by cell growth and differentiation potential. Human fibroblasts were treated with AF-MSC-CM and analyzed for cell signaling changes. For in vivo wound healing assay, ICR mice with a full-thickness skin wound were used. RESULTS: Selenium played a critical role in in vitro expansion of AF-MSCs through activation of the AKT-ERK1/2, Smad2, and Stat3 signaling pathways along with inactivation of GSK3ß. When administered together with bFGF, it showed remarkable effect in inhibiting ROS accumulation and preserving their multipotency. Proliferation and migration of human dermal fibroblasts and in vivo wound healing were improved in the CMs derived from AF-MSCs exposed to selenium and bFGF, which was caused by the Smad2, AKT-MEK1/2-ERK, and NFκB signaling triggered by the paracrine factors of AF-MSCs, such as TGF-ß, VEGF, and IL-6. Our results suggest the following: (a) supplementation of selenium in AF-MSC culture contributes to in vitro expansion and preservation of multipotency, (b) ROS accumulation causes progressive losses in proliferative and differentiation potential, (c) the separate activities of bFGF and selenium in MSCs exert an additive effect when used together, and (d) the additive combination improves the therapeutic effects of AF-MSC-derived CMs on tissue repair and regeneration. CONCLUSION: Antioxidants, such as selenium, should be considered as an essential supplement for eliciting the paracrine effects of MSC-CMs.

Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells.

Human embryonic stem cells (hESCs) need feeder cells for their maintenance in an undifferentiated state. In conventional culture systems, mouse embryonic fibroblasts (MEFs) serve as feeder cells to maintain hESCs. However, the use of MEFs elevates the risk of transmitting mouse pathogens and thus limits the potential of hESCs in cell replacement therapy. Consequently, the use of human feeder cells would be an important step forward in this in vitro technology. To address this issue, we used fibroblast-like cells differentiated from the Miz-hES6 hESC line (DiffMiz-hES6) as feeder cells to support the in vitro growth of three hESC lines. Immunofluorescence microscopy and reverse transcription-PCR assessing the expression of undifferentiated hESC markers revealed all three hESC lines were maintained in an undifferentiated state. In vitro proliferation proceeded as efficiently as when the hESCs were cultured on MEFS. Moreover, karyotype analysis revealed the chromosomal normality of the hESC lines and the DiffMiz-hES6 feeders themselves after even 50 passages. Furthermore, the hESC lines maintained their pluripotency since they remained capable of forming embryoid bodies (EBs) in vitro. Thus, hESC-derived fibroblast-like cells successfully support in vitro hESC propagation.

Efficient derivation of new human embryonic stem cell lines.

Human embryonic stem (hES) cells, unlike most cells derived from adult or fetal human tissues, represent a potentially unlimited source of various cell types for basic clinical research. To meet the increased demand for characterized hES cell lines, we established and characterized nine new lines obtained from frozen-thawed pronucleus-stage embryos. In addition, we improved the derivation efficiency from inner cell masses (to 47.4%) and optimized culture conditions for undifferentiated hES cells. After these cell lines had been maintained for over a year in vitro, they were characterized comprehensively for expression of markers of undifferentiated hES cells, karyotype, and in vitro/in vivo differentiation capacity. All of the cell lines were pluripotent, and one cell line was trisomic for chromosome 3. Improved culture techniques for hES cells should make them a good source for diverse applications in regenerative medicine, but further investigation is needed of their basic biology.

Conversion of mouse fibroblasts into cardiomyocyte-like cells using small molecule treatments.

The possibility of controlling cell fates by overexpressing specific transcription factors has led to numerous studies in stem cell research. Small molecules can be used, instead of transcription factors, to induce the de-differentiation of somatic cells or to induce pluripotent cells (iPSCs). Here we reported that combinations of small molecules could convert mouse fibroblasts into cardiomyocyte-like cell without requiring transcription factor expression. Treatment with specific combinations of small molecules that are enhancer for iPSC induction converted mouse fibroblasts into spontaneously contracting, cardiac troponin T-positive, cardiomyocyte-like cells. We specifically identified five small molecules that can induce mouse fibroblasts to form these cardiomyocyte-like cells. These cells are similar to primary cardiomyocytes in terms of marker gene expression, epigenetic status of cardiac-specific genes, and subcellular structure. Our findings indicate that lineage conversion can be induced not only by transcription factors, but also by small molecules.

Reprogramming of mouse somatic cells into pluripotent stem-like cells using a combination of small molecules.

Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) by overexpression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc. However, exogenous expression of pluripotency factors raised concerns for clinical applications. Here, we show that iPS-like cells (iPSLCs) were generated from mouse somatic cells in two steps with small molecule compounds. In the first step, stable intermediate cells were generated from mouse astrocytes by Bmi1. These cells called induced epiblast stem cell (EpiSC)-like cells (iEpiSCLCs) are similar to EpiSCs in terms of expression of specific markers, epigenetic state, and ability to differentiate into three germ layers. In the second step, treatment with MEK/ERK and GSK3 pathway inhibitors in the presence of leukemia inhibitory factor resulted in conversion of iEpiSCLCs into iPSLCs that were similar to mESCs, suggesting that Bmi1 is sufficient to reprogram astrocytes to partially reprogrammed pluripotency. Next, Bmi1 function was replaced with Shh activators (oxysterol and purmorphamine), which demonstrating that combinations of small molecules can compensate for reprogramming factors and are sufficient to directly reprogram mouse somatic cells into iPSLCs. The chemically induced pluripotent stem cell-like cells (ciPSLCs) showed similar gene expression profiles, epigenetic status, and differentiation potentials to mESCs.

Differentiation of human labia minora dermis-derived fibroblasts into insulin-producing cells.

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin- producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis- derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal- endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic ß-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin- induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.