Semi-Solid Decalcification and Research System: a Novel Method to Study Fluorescence Protein Gene Modified Stem Cells In Bone.

It remains a huge challenge to observe fluorescent protein (GFP, RFP, etc.) gene-marked cells in bone, since bone is compact, poor lucency and porous, with different tissues such as vessels, nerve, cells, matrix and blood interlacing inside. However, it is very important to study the location, growth, migration and interaction of different stem cells and their offspring in bone.

To better study different fluorescent protein gene-marked stem cells and microenvironment in bone, we establish a novel semi-solid decalcification (SSD) and research system.

1)Transplantation: Male BABL/C-GFP(H-2d) transgenic mice as donors and female FVB-RFP(H-2q) transgenic mice as recipients. Each RFP recipient mice were injected i.v. 5×10e6 allogeneic GFP bone marrow cells after 8 Gy TBI (n=10). Routinely, mice survival, weight, hemogram, GVHD manifestation were observed, with the fluorescence cells in peripheral blood and organs being traced. 2)Sections preparation: Total body perfusion fixation was performed with paraformaldehyde 21d after transplantation, and then different samples were collected for pathological examinations. The femurs were made frozen sections after semi-solid decalcification (SSD) system, while GMA plastic-embedding sections without decalcification, paraffin sections after EDTA decalcification, frozen sections after EDTA decalcification were also prepared as controls. Sections were observed by confocal microscopy. 3)Other researches: After SSD, observation and three-dimensional reconstruction were done by confocal microscopy; target tissue and cells were picked up for real-time quantified PCR for fluorescent protein genes and cell proliferation cytokines.

1)Recipients RFP mice gained WBC recovery on (18.0±1.2)d, 90.0%±2.3% peripheral cells were GFP⁺ (n=10), 6 of 10 developed GVHD within 3m. 2)During SSD, hard component of the bone disappeared slowly, replaced gradually by semi-solid substance. SSD is even workable when the bone's diameter is large than 10cm. Frozen sections after SSD clearly showed unchanged position, form, and fluorescence of the GFP and RFP cells with repeatable hematoxylin and eosin(HE)and Wright-giemsa (RG) staining and immunohistochemical staining, fluorescence chromosomal in situ hybridization (FISH) after fluorescence observation and information from different tests of the same section can also be synthesized by computer. However, GMA cold embedding section could keep the cells where they are while losing the fluorescence, further more, embedding section only works well when the bone tissue is small (diameter<2mm). Frozen section after EDTA decalcification could keep the fluorescence with changed position and form during the progress. Paraffin sections can't keep neither the fluorescence nor the normal cell position and morphological characteristics. 3)Three-dimensional reconstruction shows the interesting relationships between different cells with different fluorescence and microenvironment by confocal microscopy. Quantified PCR described the cytokine expression profile of different fluorescence gene-marked cells.

The SSD system shows great potency for the research of stem cells in vivo in bone while maintaining the morphological characteristics and structures between different cells without losing fluorescence signals. Another fantastic advantage is that a large number of techniques can be combined to our system to help us understand the homing, growth, proliferation, differentiation, migration and interaction of different target stem cells and their offspring.

No relevant conflicts of interest to declare.