Methods for Stem Cell Production and Therapy

The present invention relates to methods for rapidly expanding a stem cell population with or without culture supplements in simulated microgravity conditions. The present invention relates to methods for rapidly increasing the life span of stem cell populations without culture supplements in simulated microgravity conditions. The present invention also relates to methods for increasing the sensitivity of cancer stem cells to chemotherapeutic agents by culturing the cancer stem cells under microgravity conditions and in the presence of omega-3 fatty acids. The methods of the present invention can also be used to proliferate cancer cells by culturing them in the presence of omega-3 fatty acids. The present invention also relates to methods for testing the sensitivity of cancer cells and cancer stem cells to chemotherapeutic agents by culturing the cancer cells and cancer stem cells under microgravity conditions. The methods of the present invention can also be used to produce tissue for use in transplantation by culturing stem cells or cancer stem cells under microgravity conditions. The methods of the present invention can also be used to produce cellular factors and growth factors by culturing stem cells or cancer stem cells under microgravity conditions. The methods of the present invention can also be used to produce cellular factors and growth factors to promote differentiation of cancer stem cells under microgravity conditions

Hydrofocusing Bioreactor Produces Anti-Cancer Alkaloids

A methodology for growing three-dimensional plant tissue models in a hydrodynamic focusing bioreactor (HFB) has been developed. The methodology is expected to be widely applicable, both on Earth and in outer space, as a means of growing plant cells and aggregates thereof under controlled conditions for diverse purposes, including research on effects of gravitation and other environmental factors upon plant growth and utilization of plant tissue cultures to produce drugs in quantities greater and at costs lower than those of conventional methodologies. The HFB was described in Hydro focus - ing Bioreactor for Three-Dimensional Cell Culture (MSC-22358), NASA Tech Briefs, Vol. 27, No. 3 (March 2003), page 66. To recapitulate: The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear liquid culture environment simultaneously with the herding of suspended cells and tissue assemblies and removal of unwanted air bubbles. The HFB includes a rotating cell-culture vessel with a centrally located sampling port and an internal rotating viscous spinner attached to a rotating base. The vessel and viscous spinner can be made to rotate at the same speed and direction or different speeds and directions to tailor the flow field and the associated hydrodynamic forces in the vessel in order to obtain low-shear suspension of cells and control of the locations of cells and air bubbles. For research and pharmaceutical-production applications, the HFB offers two major benefits: low shear stress, which promotes the assembly of cells into tissue-like three-dimensional constructs; and randomization of gravitational vectors relative to cells, which affects production of medicinal compounds. Presumably, apposition of plant cells in the absence of shear forces promotes cell-cell contacts, cell aggregation, and cell differentiation. Only gentle mixing is necessary for distributing nutrients and oxygen. It has been postulated that inasmuch as cells in the simulated microgravitation of an HFB do not need to maintain the same surface forces as in normal Earth gravitation, they can divert more energy sources to growth and differentiation and, perhaps, to biosynthesis of greater quantities of desired medicinal compounds. Because one can adjust the HFB to vary effective gravitation, one can also test the effects of intermediate levels of gravitation on biosynthesis of various products. The potential utility of this methodology for producing drugs was demonstrated in experiments in which sandalwood and Madagascar periwinkle cells were grown in an HFB. The conditions in the HFB were chosen to induce the cells to form into aggregate cultures that produced anti-cancer indole alkaloids in amounts greater than do comparable numbers of cells of the same species cultured according to previously known methodologies. The observations made in these experiments were interpreted as suggesting that the aggregation of the cells might be responsible for the enhancement of production of alkaloids

Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas

Rapid Selection and Proliferation of CD133(+) Cells from Cancer Cell Lines: Chemotherapeutic Implications

Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133(+)] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133(+) cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a (+)15-fold proliferation of the CD133(+) cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (−)4.8-fold decrease in the CD133(+)cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133(+) cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates

Comparison of CD133(+) and (−) cel counts ± standard deviation of SAOS-2 cells grown in the HFB or RCCS vessels.

<p>Comparison of CD133(+) and (−) cel counts ± standard deviation of SAOS-2 cells grown in the HFB or RCCS vessels.</p

Fluorescence index of various stem cell and differentiation markers examined in cells cultured in 1-G static condition, sorted by CD133 antibody or not, and after culture in the bioreactor for 5 days.

<p><b>A</b>) Diagram of the expression levels of various bio-markers. Lavender bars indicate the percentage of cells expressing basal levels of the various markers in parental SAOS-2 cells cultured in normal 1-G gravity condition (control). Purple bars indicate the percentage of cells expressing the various markers in CD133(+) MACSorted SAOS-2 cells that were isolated from parental SAOS-2 cells cultured in normal 1-G gravity condition. Yellow bars indicate the percentage of cells expressing the various markers in CD133(+) MACSorted SAOS-2 cells that were cultured in hypogravity condition. Light blue bars indicate the percentage of cells expressing the various markers in parental SAOS-2 cells that were cultured in hypogravity condition for 5-days, resulting in a population of selected and proliferated CD133(+) SAOS-2 cells. <b>B</b>) Immunofluorescence staining (40×) and positivity of SAOS-2 cells to CD133, Sox-9, SPARC, and CD117/c-Kit following growth in the bioreactor for 5-days.</p

Average number of colonies counted ± standard deviation in 6-well dishes in a triplicate experiment.

<p>Average number of colonies counted ± standard deviation in 6-well dishes in a triplicate experiment.</p

Sensitivity of SAOS-2 cells to doxorubicin following growth in simulated hypogravity.

<p><b>A</b>) LD₅₀ for doxorubicin determined for the SAOS-2 cells. An LD₅₀ of 0.5µg/mL for doxorubicin was determined exposing the SAOS-2 cells to a 24-hours treatment to the drug, using an MTT assay. Comparable results were obtained with cell count by trypan blue exclusion. <b>B</b>) Histogram showing the sensitivity of SAOS-2 cells to 0.25µg/mL of doxorubicin following a 24-hours treatment, and using an MTT assay. CD133(+) cells are sensitive to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are even more sensitive to the treatment, instead. <b>C</b>) Histogram showing the sensitivity of SAOS-2 cells to 0.5µg/mL of doxorubicin following a 24-hours treatment, using an MTT assay. CD133(+) cells show sensitivity to the clinically relevant dose of 0.5 µg/mL of doxorubicin, however the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are greatly sensitized to the chemotherapy treatment. <b>D</b>) Histogram showing the sensitivity of SAOS-2 cells to a dose of 1.1µg/mL of doxorubicin following a 24-hours treatment, and using an MTT assay. The CD133(+) cells show sensitivity to a dose of 1.1µg/mL of doxorubicin, however the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitized to the chemotherapy treatment. <b>E</b>) Histogram showing the sensitivity of SAOS-2 cells to 2.2µg/mL of doxorubicin following a 24-hours treatment, and using an MTT assay. CD133(+) cells are sensitive to the chemotherapy treatment, but the CD133(+) SAOS-2 cells proliferated and selected with the HFB culture system are sensitive, instead.</p

Phase contrast images of spheres formed by HFB SAOS-2 CD133(+) enriched osteosarcoma cells.

<p><b>A</b>) HFB grown SAOS-2 osteosarcoma cells (CD133+) are able to proliferate and assemble three-dimensionally as sarcosheres after 3-days of culture in the bioreactor (10× magnification). <b>B</b>) HFB grown SAOS-2 osteosarcoma cells (CD133+) are able to proliferate and assemble three-dimensionally as sarcosheres after 3-days of culture in the bioreactor (40× magnification). <b>C</b>) HFB grown SAOS-2 osteosarcoma cells (CD133+) seeded into an irradiated plastic culture dish, reconstitute the normal attached phenotype of the parental SAOS-2 cells after one week of culture (10× magnification). Arrows point at CD133(+) SAOS-2 cells showing a rounded and weakly attaching phenotype. <b>D</b>) HFB grown SAOS-2 osteosarcoma cells (CD133+) seeded into an irradiated plastic culture dish, reconstitute the normal attached phenotype of the parental SAOS-2 cells after one week of culture (40× magnification). Arrow points at CD133(+) SAOS-2 cells showing a rounded and weakly attaching phenotype.</p