Research Express@NCKU - Articles Digest (Volume 17 Issue 5)

http://research.ncku.edu.tw/re../articles/e/20110225/4.html

The use of nanoparticles and microfluidic system for isolation, differentiation and tracking of mesenchymal stem cells for cancer therapy
Chao-Liang Wu
Distinguished Professor, Department of Biochemistry and Molecular Biology wumolbio@mail.ncku.edu.tw NCKU Landmark ProjectR010

esenchymal stem cells (MSCs) are defined by three main criteria, including self-renewal, ability to differentiate

into multiple cell types, and capability of in vivo reconstitution of a given tissue. Multipotent MSCs isolated from bone marrow and other sites are currently being studied to determine their potential for therapeutic applications. They have been demonstrated to play a role in tissue repair and regeneration in both preclinical and clinical studies. However, the properties of MSCs are poorly understood as they are generally identified through a combination of poorly defined physical, phenotypic, and functional properties. Therefore, isolation and characterization of MSCs are needed for studying stem cell biology and for further therapeutic applications. MSCs can be isolated from bone marrow, fetus, cord blood as well as other tissues and organs.In this program project, first we propose to develop integrated microfluidic culture systems for isolation and differentiation of MSCs from cord blood and fetus bone marrow. Since their processes of self-renewal and differentiation into multiple lineages are tightly regulated by genetic and epigenetic mechanisms, identification and understanding of the epigenetic signatures during differentiation are critical for classifying differentiating cells and understanding molecular networks active during such progression. For tracking and therapeutic purposes, we also attempt to develop nanoparticle-assisted photo/thermo therapy by NIR irraditation. MSCs, when transplanted systemically, can selectively migrate to and proliferate in solid tumors. Systemic administration of modified MSCs targeting to multiple tumor sites showed antitumor effects in animal tumor models, suggesting that MSCs may be used as a cellular vehicle of gene delivery to tumor sites. Oncolytic adenoviruses are attractive therapeutics for cancer because they selectively replicate in tumors and cause oncolysis. However, the barriers that limit its therapeutic efficacy include poor transduction of tumor cells and inefficient systematic delivery to tumor sites. We hypothesize that the tumor-homing properties of MSCs can effectively enhance systemic delivery of oncolytic adenoviruses to metastatic tumor sites. In this study, we also used MSCs to carry two oncolytic adenoviruses that we have developed to target tumors that express Oct-3/4, an embryonic stem cell and tumor marker, for the treatment of metastatic tumors. The results of this study are shown below: Culturing and differentiation of mesenchymal stem cells by using a microfluidic system A microfluidic system with several integrated components, including stem cell culture areas, micropumps, microgates, seeding reservoirs, waste reservoirs and fluid microchannels, was developed for culturing, and differentiation of mesenchymal stem cells (MSC) (Fig. 1). The developed automated system allows for the long-term culture and differentiation of MSCs. Experimental results clearly demonstrate that the MSCs can be cultured for proliferation and different types of differentiation are possible implemented in this microfluidic system, which can maintain a suitable and stable pH value over long time periods. This prototyped microfluidic system has great potential as a powerful tool for future MSC studies.

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Research Express@NCKU - Articles Digest (Volume 17 Issue 5)

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Figure 1. Schematic representation of the automatic microfluidic system for the culture and differentiation of stem cells .

An integrated microfluidic system for isolation, counting and sorting of hematopoietic stem cells An integrated microfluidic system was developed for isolation, counting and sorting of hematopoietic stem cells (HSCs) from cord blood in an automatic format by utilizing a magnetic bead-based immunoassay . Three functional modules including a cell isolation module, a cell counting module and a cell sorting module are integrated on a single chip by using microfluidic technology (Fig. 2). It is composed of a four-membrane-type micromixer for binding of target stem cells and magnetic beads, two pneumatic micropumps for sample and reagent transport, and an S-shaped channel for isolation of HSCs using a permanent magnet underneath. After the incubation process, the cell counting and sorting modules was used for HSCs counting and sorting. The results show that a separation efficiency as high as 88 % for HSCs is achieved within 40 minutes. This integrated microfluidic system may be promising for stem cells applications.

Figure 2 (a) Schematic illustration of procedure performed on the microfluidic chip for HSC detection including a cell isolation module, a cell counting module and a cell sorting module; (b) An illustration of the integrated microfluidic chip including several elements .

Development of nanoparticle-assisted photo/thermo therapy by NIR irraditation For uses of Au nanoparticles in photothermal imaging and the photothermal treatment of tumor, it is more convenient to shift the surface palsmon resonance band to the red/NIR region where the tissue light absorbed fraction is negligible. Lanthanum hexabromide (LaB6) NPs exhibited a strong absorption arising from the surface plasmon resonance and have a higher photothermal conversion efficiency than gold nanoparticles and nanoshells in the NIR region. Because of low price, it may be used as an alternate of gold nanomaterials. In this work, well-dispersed LaB6@SiO2@Au NPs have been successfully fabricated by the milling of LaB6 particles, sol-gel coating of SiO2 (i.e., LaB6@SiO2 NPs), surface modification with (3-Aminopropyl)triethoxysilane (APTES), attachment of gold seeds (i.e., LaB6@SiO2/Au NPs), and the followed coating of gold nanoshells via the reduction of HAuCl4 (Fig. 3). They exhibited significant absorption in the VIS/NIR region and excellent photothermal conversion ability under the

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Research Express@NCKU - Articles Digest (Volume 17 Issue 5)

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NIR irradiation (Fig. 4). Such a novel nanocpmposite is expected to be useful in photothermal therapy.

Figure 3. TEM images of LaB6 NPs (a), LaB6@SiO2 NPs (b), LaB6@SiO2/Au NPs (c), and the LaB6@SiO2@Au NPs obtained in the HAuCl4 solutions of 0.135 (d), 0.27 (e) and 0.54 (f) mM. This figure reveals that LaB6 NPs were well dispersed. After SiO2 coating, they remained discrete.

Figure 4. Variations of temperature with the irradiation time for LaB6@SiO2 NPs, LaB6@SiO2/Au NPs, and the LaB6@SiO2@Au NPs obtained in the HAuCl4 solutions of 0.135, 0.27 and 0.54 mM. For the cases of LaB6@SiO2@Au NPs, the temperature increase became more significant with the increase of HAuCl4 concentration because the coverage of gold nanoshells was more complete.

Isolation and tumor-trophic characterization of MSCs. Mice bone marrow-derived mesenchymal stem cells (MSCs) were isolated. The abilities of MSCs to differentiate into adipocytes and osteocytes were demonstrated by the presence of Oil Red-O staining and Alkaline Phosphatase staining, respectively (Fig. 5A). Using a transwell migration assay, MSCs plated on the upper chamber migrated toward tumor from tumor conditioned medium or MSCs conditioned medium in the lower chamber. Figure 5B shows results of typical migration assays after 8 hours. We were able to observe a significant increase in migration of MSCs after were exposure to conditioned medium from tumor cells as compared with to MSCs conditioned medium.

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Research Express@NCKU - Articles Digest (Volume 17 Issue 5)

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Figure 5. Isolation and tumor-trophic characterization of MSCs. (a) Differentiation potential of mesenchymal stem cells (MSCs). (b) Tumor conditioned medium were placed in the lower chamber and the membrane was stained for the presence of MSCs after 8 hours of migration as compared with control containing MSCs conditioned medium.

Systemically administered MSCs-AdGS2 reduces the growth of MBT-2 cell derived lung metastases in vivo. We investigated the in vivo anti-tumor activity of MSCs-AdGS2. We injected MBT-2 cells intrapleural into the thoracic cavity of immunocompetence mice to establish pulmonary metastases. Four days later, we injected 1X106 MSCs loaded with Ad-GS2 or AdLuc intravenous. Control mice received intravenous injection of saline. Eighteen days after tumor cell injection, the mice were sacrificed, and the weights of whole lungs and tumor were measured. H&E showing representative lung metastases postintrapleural injection of MBT-2 cells into C3H/HeN. Mice intravenously injected with MSCs-AdGS2 had smaller tumor nodule than MSCs-AdLuc treated or control saline treated mice (Fig.6A). As shown in Fig.6B, Mice injected with tumor cells and intravenously with MSCs-AdGS2 had smaller mean lung and tumor weight than MSCs-AdLuc treated mice or control saline treated mice (P < 0.05). Mice were treated on day 4 after intrapleural injection with MBT-2 with one dose of intravenous injection of MSCs-AdGS2, MSCs-AdLuc, or saline treatment. Immunostained against Luciferase and Ad hexon (as an indicator of viral replication) were performed to identify cell populations in lung sections (Fig.6C). For evaluating the antitumor efficacy another lung metastatic animal model to investigate, MBT-2 cells (10 5) were inoculated i.v. into the C3H/HeN mice at day 0. At day 7, four groups of 6 mice were treated i.v. with 109 plaque-forming units (PFU) of MSC-AdGS2, MSC-AdLuc, AdGS2, or with saline. MSC-AdGS2 treatment also significantly prolonged the survival time of the tumor-bearing mice compared with AdGS2, MSC-AdLuc, or saline treatment (Fig.6D). In mice treated with MSC-AdGS2, 40 % survived by 80 days, while the survival rate was 0 % in other groups.

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Research Express@NCKU - Articles Digest (Volume 17 Issue 5)

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Figure 6. The antitumor effects of oncolytic adenoviruses-loaded MSCs in animal tumor models. oncolytic adenoviruses-loaded MSCs reduce the growth and prolong survival of lung metastases.

Summary During 2-year project, some greatly significant achievements were produced. For a MSC culture system, an automatic microfluidic chip capable of culturing and differentiating stem cells for a long period of time was successfully demonstrated. Moreover, a simple and efficient method for isolation, counting and sorting of hematopoietic stem cells was realized by using a microfluidic device. The separation efficiency is even superior to the conventional method. Furthermore, well-dispersed LaB6@SiO2@Au NPs have been successfully fabricated by the milling of LaB6 particles, sol-gel coating of SiO2, surface modification with APTES, attachment of gold seeds, and the followed coating of gold nanoshells. They exhibited strong absorption in the VIS/NIR region and excellent photothermal conversion ability under the NIR irradiation. It is potential to develop as a reagent for photo-thermal therapy. In addition, we have also established MSC to carry oncolytic adenoviruses to enhance their therapeutic efficacy through their homing properties to tumor tissues.

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