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Personalized Stem Cells by Stemgenics
StemgenicsTM

Personalized Stem Cells

Technology

Stemgenics Technology

The Stemgenics proprietary technology reprograms mature cells into stem cells.

Functionalized nanoparticles are used to carry reprogramming elements into the nucleus where they interact with DNA to reprogram the cells.

Experiments show that functionalized nanoparticles enter the cytoplasm and the nucleus of cells and reprogram mature human cells into stem cells.

The stem cells have been characterized as pluripotent stem cells by growth, morphology, stem cell characteristic transcription patterns, and alkaline phosphatase expression.

Nanoparticles targeted to cell cytoplasm Figure 2. Nanoparticles targeted to cell cytoplasm. Two different types of Human cells (A) and (B) were treated for 30 minutes with fluorescent nanoparticles functionalized to target cell cytoplasm, washed and stained with DAPI (blue fluorescence showing nuclei). The green fluorescence shows concentration of the nanoparticles in the cell cytoplasm.

Imaging of human cells treated with nanoparticles functionalized with a fluorescent dye and cytoplasmic targeting elements showed that functionalized nanoparticles effectively enter the cytoplasm of the cells. Representative high resolution images of the cells with cytoplasmic localization of functionalized nanoparticles are depicted in Figure 2.

Nuclear localization of nanoparticle functionalized with cytoplasmic and nuclear targeting elements Figure 3. Nuclear localization of nanoparticle functionalized with cytoplasmic and nuclear targeting elements (nanoparticles-CN). After 26 hours of treatment of human fibroblasts with fluorescently labeled nanoparticles-CN, live cells were washed and visualized by A-phase contrast microscopy and B-fluorescent microscopy. The fluorescence shows nanoparticles concentrated in the nuclei of treated cells.

Fluorescently (FITC) labeled nanoparticles functionalized with cell reprogramming small and larger molecules, cytoplasmic and nuclear targeting elements effectively enter the cells and concentrate in the nucleus of treated cells. Representative high resolution images of live cells with fluorescently labeled nanoparticles are depicted in Figure 3.

Functionalized nanoparticles induce expression of various target genes in human cells. Figure 4. Functionalized nanoparticles induce expression of various target genes in human cells.

To demonstrate that functionalized nanoparticles not only enter the nucleus but also modify target gene expression, real-time RT-PCR analysis was used to analyze gene expression of actin and four target genes in control cells and cells treated with functionalized nanoparticles. The real-time RT-PCR results depicted in Figure 4 demonstrate that while actin expression was unchanged, the target genes were overexpressed 58, 8, 22, and 57 fold.

Alkaline phosphatase staining of stem cells reprogramed with functionalized superparamagnetic nanoparticles Figure 5. Reprogramming somatic cells into stem cells. The left panel shows fibroblast cells (skin cells) in culture: long narrow cells. The panel on the right shows reprogrammed stem cells: an early stage colony.

Culturing mature human cells with cell reprogramming nanoparticles functionalized with bioactive molecules produces early stage stem cell colonies of 100 microns as shown in Figure 5 and late stage stem cell colonies of 200 microns as shown in Figure 6.

Alkaline phosphatase staining of stem cells reprogramed with functionalized superparamagnetic nanoparticles Figure 6. Reprogramming somatic cells into stem cells. The left panel shows fibroblast cells (skin cells) in culture: long narrow cells. The panel on the right shows reprogrammed stem cells: a late stage colony of 200 microns.

Cells reprogrammed from mature human cells into pluripotent stem cells have been characterized by growth, morphology, and patterns of transcription by real-time qPCR analysis. The change in transcription patterns from mature human cells to those typical of pluripotent stem cells has been observed.

Alkaline phosphatase staining of stem cells reprogramed with functionalized nanoparticles Figure 7. Alkaline phosphatase staining of stem cells reprogramed with functionalized nanoparticles. Note green fluorescence of colony from fluorescent alkaline phosphatase substrate (preliminary data).

The reprogrammed stem cell-like colonies express alkaline phosphatase as expected of pluripotent stem cells. Note the alkaline phosphatase assay results depicted in Figure 7.