Shiaw-min Hwang - Academia.edu (original) (raw)

Papers by Shiaw-min Hwang

Nucleic Acids Research

Baculovirus (BV) is a promising gene vector but mediates transient expression. To prolong the exp... more Baculovirus (BV) is a promising gene vector but mediates transient expression. To prolong the expression, we developed a binary system whereby the transgene in the substrate BV was excised by the recombinase (ΦC31o, Cre or FLPo) expressed by a second BV and recombined into smaller minicircle. The recombination efficiency was lower by ΦC31o (≈40-75%), but approached ≈90-95% by Cre and FLPo in various cell lines and stem cells [e.g. human adipose-derived stem cells (hASCs)]. Compared with FLPo, Cre exerted higher expression level and lower negative effects; thus, we incorporated additional cis-acting element [oriP/Epstein-Barr virus nuclear antigen 1 (EBNA1), scaffold/matrix attached region or human origin of replication (ori)] into the Cre-based BV system. In proliferating cells, only oriP/EBNA1 prolonged the transgene expression and maintained the episomal minicircles for 30 days without inadvertent integration, whereas BV genome was degraded in 10 days. When delivering bmp2 or vegf...

Repair of large calvarial bony defect remains a challenge for orthopedic surgeons. Since microRNA... more Repair of large calvarial bony defect remains a challenge for orthopedic surgeons. Since microRNAs (miRNAs) modulate the osteogenesis of osteoprogenitor cells, we aimed to engineer human adiposederived stem cells (hASCs), a promising cell source for bone engineering, with miRNA-expressing baculovirus vectors. We constructed 4 baculoviruses each expressing 1 human miRNA (miR-26a, miR-29b, miR-148b, miR-196a) and verified that the miRNA-expressing baculovirus vectors augmented hASCs osteogenesis. Among these 4 miRNAs, miR-148b and miR-196a exerted more potent osteoinductive effects than miR-26a and miR-29b. Furthermore, we unveiled that co-transduction of hASCs with miR-148b-expressing and bone morphogenetic protein 2 (BMP-2)-expressing baculovirus vectors enhanced and prolonged BMP-2 expression, and synergistically promoted the in vitro osteogenic differentiation of hASCs. Implantation of the hASCs co-expressing BMP-2/miR-148b into critical-size (4 mm in diameter) calvarial bone defects in nude mice accelerated and potentiated the bone healing and remodeling, filling z94% of defect area and z89% of defect volume with native calvaria-like flat bone in 12 weeks, as judged from micro computed tomography, histology and immunohistochemical staining. Altogether, this study confirmed the feasibility of combining miRNA and growth factor expression for synergistic stimulation of in vitro osteogenesis and in vivo calvarial bone healing.

Biotechnology and Bioengineering

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem... more We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs) and MSCs-derived adipogenic, chondrogenic, and osteogenic progenitors without compromising the differentiation capacity. Remarkably, the transgene expression level and duration varied widely with the differentiation states at which the progenitors were transduced. However, whether the variation was a general phenomenon and what caused the variation were unclear. Here we demonstrated that transduction of the MSCs and MSC-derived progenitors using baculoviruses carrying egfp driven by CMV, EF-1alpha or CAG promoter resulted in a general trend of varied expression, that is, the chondrogenic progenitors allowed for the poorest expression while the adipogenic progenitors conferred the best expression. Quantification of the nuclear and cytoplasmic egfp gene copy numbers by quantitative real-time PCR revealed that the varied expression did not arise from the discrepancies in gene ...

Scientific reports, 2015

It is desirable to obtain unlimited supplies of endothelial cells for research and therapeutics. ... more It is desirable to obtain unlimited supplies of endothelial cells for research and therapeutics. However, current methods of deriving endothelial cells from humans suffer from issues, such as limited supplies, contamination from animal substances, and lengthy/complicated procedures. In this article we developed a way to differentiate human iPS and ES cells to highly pure endothelial cells in 5 days. The chemically defined system is robust, easy to perform, and free of animal substances. Using the system, we verified that combined TGFβ and canonical Wnt agonists are essential and sufficient for iPS/ES cell-to-mesoderm transition. Besides, VEGF-KDR signaling alone is required for endothelial formation at high density while supplementation with FGF allows for colonial endothelial differentiation. Finally, anti-adsorptive agents could enrich the endothelial output by allowing selective attachment of the endothelial precursors. The system was validated to work on multiple iPS/ES cells li...

Biomaterials, 2015

Long non-coding RNAs (lncRNAs) play regulatory roles in cancers. LncRNA PTENP1 is a pseudogene of... more Long non-coding RNAs (lncRNAs) play regulatory roles in cancers. LncRNA PTENP1 is a pseudogene of the tumor suppressor gene PTEN but its roles in hepatocellular carcinoma (HCC) have yet to be explored. Here we confirmed that PTENP1 and PTEN were downregulated in several HCC cells, thus we constructed Sleeping Beauty (SB)-based hybrid baculovirus (BV) vectors for sustained PTENP1 lncRNA expression. Co-transduction of HCC cells with the SB-BV vector expressing PTENP1 elevated the levels of PTENP1 and PTEN, which suppressed the oncogenic PI3K/AKT pathway, inhibited cell proliferation, migration/invasion as well as induced autophagy and apoptosis. The overexpressed PTENP1 decoyed oncomirs miR-17, miR-19b and miR-20a, which would otherwise target PTEN, PHLPP (a negative AKT regulator) and such autophagy genes as ULK1, ATG7 and p62, indicating that PTENP1 modulated the HCC cell behavior and gene networks by miRNA regulation. Injection of the PTENP1-expressing SB-BV vector into mice bearin...

Taiwanese journal of obstetrics & gynecology, 2012

To test the hypothesis that human amniotic fluid mesenchymal stem cells contain a unique epigenet... more To test the hypothesis that human amniotic fluid mesenchymal stem cells contain a unique epigenetic signature in imprinting centers of H19, SNRPN, and KCNQ1OT1 during in vitro cell culture. By bisulfite genomic sequencing, we analyzed the imprinting centers of three imprinted genes (including H19, SNRPN, and KCNQ1OT/) in a total of six single-cell clones of human amniotic fluid mesenchymal stem cells at cell passages 7, 8, 9, and 10 during in vitro cell culture. The imprinting centers of H19 and KCNQ1OT1 showed hypermethylation at passage 7 in all single-cell clones of human amniotic fluid mesenchymal stem cells, and there was no significant change in DNA methylation patterns during in vitro cell culture. The imprinting centers of SNRPN showed variable methylation patterns at passage 7 in six single-cell clones, and DNA methylation patterns varied during in vitro cell culture from passages 8 to 10. In conclusion, human amniotic fluid mesenchymal stem cells contain a unique epigeneti...

Methods in molecular biology (Clifton, N.J.), 2012

Human mesenchymal stromal cells (hMSCs) play a crucial role in tissue engineering and regenerativ... more Human mesenchymal stromal cells (hMSCs) play a crucial role in tissue engineering and regenerative medicine and thus have important clinical potential for cell-based therapy. However, the limited cell number and the difficulty in detecting these cells in vivo have restricted many hMSC studies. Therefore, the development of hMSCs immortalized with telomerase and expressing red fluorescence protein will facilitate their expansion and detection in vivo, and these cells will be important for both to stem cell research and clinical use. In this chapter, we describe the protocols used to establish telomerase- and red fluorescence protein-expressing immortalized hMSCs using a nonviral transfection method. These cells will be useful tools for stem cell research and translational studies.

Methods in molecular biology (Clifton, N.J.), 2011

We have used Affymetrix oligonucleotide microarrays to analyze common transcriptomes and thereby ... more We have used Affymetrix oligonucleotide microarrays to analyze common transcriptomes and thereby learn about the core gene expression profile in human mesenchymal stem cells (MSC) from different tissues, including fetal amniotic fluid-derived MSC, term pregnancy amniotic membrane-derived MSC, term pregnancy umbilical cord blood-derived MSC, and adult bone marrow-derived MSC. The beauty of microarray analysis of gene expression (MAGE) is that it can be used to discover associating genes that were previously thought to be unrelated to a physiological or pathological event. However, interpreting complex biological processes from gene expression profiles often requires extensive knowledge mining in biomedical literature. In this chapter, we describe, step-by-step, how to use a commercially available biological database and software program, MetaCore (GeneGo Inc.), for functional network analysis.

Methods in molecular biology (Clifton, N.J.), 2007

Human cord blood (CB), collected from the postpartum placenta and umbilical cord, has been identi... more Human cord blood (CB), collected from the postpartum placenta and umbilical cord, has been identified as a rich source of hematopoietic stem cells (HSCs) and provides an alternative to bone marrow or mobilized peripheral blood transplantation. However, the major restriction of CB transplantation is the low number of HSCs in each CB unit and limits its use in clinical transplantation. The development of ex vivo culture systems that facilitate the expansion of HSCs is crucial to stem cell research and clinical application. In this chapter, we describe the protocols to isolate HSCs from CB, expand HSCs in serum-free condition, and analyze HSCs. This information is benefical for successful use of CB stem cells in therapeutic studies.

Blood, 1994

Nitric oxide is a short-lived reactive mediator that inhibits bone marrow (BM) cell proliferation... more Nitric oxide is a short-lived reactive mediator that inhibits bone marrow (BM) cell proliferation induced by granulocyte-macrophage colony-stimulating factor (GM-CSF). The present studies show that nitric oxide also inhibits macrophage colony-stimulating factor (M-CSF)-induced growth of mouse BM cells, an effect that was dependent on the presence of an inflammatory mediator and blocked by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (L-NMA). Treatment of mice with the hematotoxicant benzene (800 mg/kg, intraperitoneally, two times per day, for 2 days) resulted in a significant increase in nitric oxide production by BM cells stimulated with lipopolysaccharide (LPS) and interferon gamma alone or in combination with M-CSF or GM-CSF. Cells from benzene-treated mice also displayed increased sensitivity to the growth-promoting effects of M-CSF and GM-CSF. These results suggest that benzene treatment of mice primes BM cells to inducers of nitric oxide. Northern blot analys...

Gaoxiong yi xue ke xue za zhi = The Kaohsiung journal of medical sciences, 1995

We have used PCR to amplify a polymorphic portion of the X-chromosome linked phosphoglycerate kin... more We have used PCR to amplify a polymorphic portion of the X-chromosome linked phosphoglycerate kinase gene (PGK) combined with a methylation-sensitive restriction enzyme digestion of the active X chromosome to examine the frequency of heterozygosity in Taiwanese females and analyze clonality in 18 female patients with acute myeloid leukemia (AML). We used hair follicles as normal tissue control. We found that the incidence of heterozygosity of the PGK gene in 102 hematological normal females and 18 patients tested was 35% (42/120). In five AML patients, a monoclonal X-inactivation pattern of leukemic blasts was found at presentation, which then returned to polyclonal at remission. In two of these five cases, a monoclonal pattern recurred at relapse. We also found that the hair follicles were readily accessible normal tissue for control, were easy to obtain, and were non-invasive.

http://www.StemCells.com the World Wide Web at:

Bone marrow and umbilical cord blood are reported to be the main sources of mesenchymal stem cell... more Bone marrow and umbilical cord blood are reported to be the main sources of mesenchymal stem cells (MSCs), which have been proposed for many clinical applications. This study evaluated and quantitated the differentiation potential of bone marrow-derived MSCs (bmMSCs) and cord blood-derived MSCs (cbMSCs) by in vitro induction. Results indicated that cbMSCs had a significantly stronger osteogenic potential but lower capacity for adipogenic differentiation than bmMSCs. Leptin, an important regulator of mesenchymal differentiation, has a significantly stronger effect of promoting osteogenesis and inhibiting adipogenesis in bmMSCs than in cbMSCs. Moreover, Cbfa1 mRNA expression in bmMSCs and cbMSCs was affected to different degrees by leptin during osteogenesis. In contrast, leptin reduced PPARgamma2 mRNA expression to the same level during adipogenesis in both types of MSCs. These results demonstrate the disparate capacities of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. In addition, the disparate differentiation tendencies of MSCs from different sources should be considered in further applications.

Molecular Therapy, 2014

MicroRNA 122 (miR-122) is a tumor suppressor for hepatocellular carcinoma (HCC) but is lowly expr... more MicroRNA 122 (miR-122) is a tumor suppressor for hepatocellular carcinoma (HCC) but is lowly expressed in HCC cells. MiR-151 is aberrantly overexpressed in HCC cells and promotes HCC metastasis yet its roles on HCC tumorigenicity are unknown. To combat HCC tumorigenicity/metastasis, we developed Sleeping Beauty (SB)-based hybrid baculovirus (BV) vectors that expressed (i) miR-122 precursors (pre-miR-122), (ii) miR-151 sponges, or (iii) pre-miR-122 and miR-151 sponges. Transduction of aggressive HCC cells (Mahlavu) with the pre-miR-122-expressing BV tremendously enhanced miR-122 levels for &amp;amp;amp;amp;amp;amp;amp;gt;6 weeks, suppressed the levels of downstream effectors (e.g., ADAM10 and Bcl-w), proliferation, anchorage-independent growth, motility and migration/invasion in vitro. Intratumoral injection of the pre-miR-122-expressing BV attenuated the HCC growth/metastasis. The miR-151 sponges-expressing BV diminished the miR-151 levels for 6 weeks, enhanced RhoGDIA expression, suppressed RhoGTPases, as well as motility and migration/invasion of Mahlavu cells. Intratumoral injection of the miR-151 sponge-expressing BV impeded not only HCC metastasis but also cell proliferation, MMP expression and tumor growth in vivo. The BV co-expressing pre-miR-122 and miR-151 sponges also simultaneously enhanced miR-122 expression and inhibited miR-151, and conferred antitumor/anti-metastasis effects albeit lack of synergism. These data implicate the potentials of the SB-based hybrid BV for persistently modulating miRNA and suppressing HCC tumorigenicity/metastasis.

Tissue Engineering Part A, 2015

We recently developed hybrid baculovirus (BV) vectors that exploited FLPo/Frt-mediated DNA minici... more We recently developed hybrid baculovirus (BV) vectors that exploited FLPo/Frt-mediated DNA minicircle formation. Engineering of adipose-derived stem cells (ASCs) with the FLPo/Frt-based BV vectors enabled prolonged transgene expression and, after cell implantation into rabbits, ameliorated cartilage regeneration and bone repair. To translate the hybrid BV one step further toward clinical applications, here we assessed the biosafety profiles of the hybrid BV-engineered human ASCs (hASCs) in vitro and evaluated the immune responses elicited by the engineered porcine ASCs (pASCs) in large animals. We confirmed that the hybrid BV did not compromise the hASCs viability, immunosuppressive capacity, and surface characteristics. Neither did the hybrid BV cause chromosomal abnormality/transgene integration in vitro nor did it induce tumorigenicity in vivo. In the large animal study, pASCs were engineered with the hybrid BV expressing bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) and implanted into femoral bone defects in mini pigs. The hybrid BV-engineered pASCs enabled prolonged BMP2/VEGF expression and triggered the healing of massive segmental bone defects, while only eliciting transient antibody, cytokine, and local cellular immune responses stemming from the implantation procedure itself. These data altogether demonstrated the safety of the hybrid BV vectors for ASCs engineering and bone healing in large animals, hence implicating the potential in clinical applications.

Scientific Reports, 2015

Down syndrome (DS) patients with early-onset dementia share similar neurodegenerative features wi... more Down syndrome (DS) patients with early-onset dementia share similar neurodegenerative features with Alzheimer's disease (AD). To recapitulate the AD cell model, DS induced pluripotent stem cells (DS-iPSCs), reprogrammed from mesenchymal stem cells in amniotic fluid, were directed toward a neuronal lineage. Neuroepithelial precursor cells with high purity and forebrain characteristics were robustly generated on day 10 (D10) of differentiation. Accumulated amyloid deposits, Tau protein hyperphosphorylation and Tau intracellular redistribution emerged rapidly in DS neurons within 45 days but not in normal embryonic stem cell-derived neurons. N-butylidenephthalide (Bdph), a major phthalide ingredient of Angelica sinensis, was emulsified by pluronic F127 to reduce its cellular toxicity and promote canonical Wnt signaling. Interestingly, we found that F127-Bdph showed significant therapeutic effects in reducing secreted Ab40 deposits, the total Tau level and the hyperphosphorylated status of Tau in DS neurons. Taken together, DS-iPSC derived neural cells can serve as an ideal cellular model of DS and AD and have potential for high-throughput screening of candidate drugs. We also suggest that Bdph may benefit DS or AD treatment by scavenging Ab aggregates and neurofibrillary tangles.

PLoS ONE, 2014

Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here,... more Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here, we explored the underlying molecular mechanisms through which Wnt signaling regulates neurotrophins (NTs) in the NT-induced neuronal differentiation of human mesenchymal stem cells (hMSCs). NTs can increase the expression of Wnt1 and Wnt7a in hMSCs. However, only Wnt7a enables the expression of synapsin-1, a synaptic marker in mature neurons, to be induced and triggers the formation of cholinergic and dopaminergic neurons. Human recombinant (hr)Wnt7a and general neuron makers were positively correlated in a dose-and time-dependent manner. In addition, the expression of synaptic markers and neurites was induced by Wnt7a and lithium, a glycogen synthase kinase-3b inhibitor, in the NT-induced hMSCs via the canonical/bcatenin pathway, but was inhibited by Wnt inhibitors and frizzled-5 (Frz5) blocking antibodies. In addition, hrWnt7a triggered the formation of cholinergic and dopaminergic neurons via the non-canonical/c-jun N-terminal kinase (JNK) pathway, and the formation of these neurons was inhibited by a JNK inhibitor and Frz9 blocking antibodies. In conclusion, hrWnt7a enhances the synthesis of synapse and facilitates neuronal differentiation in hMSCS through various Frz receptors. These mechanisms may be employed widely in the transdifferentiation of other adult stem cells.

Aims Cell transplantation is a promising approach for patients with myocardial infarction. Howeve... more Aims Cell transplantation is a promising approach for patients with myocardial infarction. However, fol- lowing injection, retention of the transplanted cells in the injected area remains a central issue, which can be deleterious to cell transplantation therapy. We hypothesized that the use of cell sheet frag- ments, with the preservation of extracellular matrix (ECM), may significantly increase cell retention and