Toward an ideal animal model to trace donor cell fates after stem cell therapy: Production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene1

Abstract

The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

Comparative immunophenotyping of equine multipotent mesenchymal stromal cells: An approach toward a standardized definition

“…CD 73 protein expression could also be detected in bone marrow derived MSCs from mice but only in moderate amounts (61). MSCs isolated from different tissues of pigs robustly express CD 90 (54,(62)(63)(64) and CD 105 (54). CD 73 investigation in pig is hampered by a lack of cross-reactive mAbs (63).…”

Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene

“…The transgenic pigs were generated by using procedures described previously [4]. Sexually mature crossbred gilts (Duroc ×Landrace × Yorkshire) aged between 240 to 300 d and weighing greater than 120 kg were used as embryo donors or recipients.…”

“…Genomic DNA was extracted as described previously [4]. A PCR analysis of the genomic DNA was performed by primers specific for DsRed (forward, 5′-ATGGACAACACCGAGGACGTCATC-3′, and reverse, 5′- TCAGTGGTATTTGTGAGCCAGGGC-3′).…”

“…In contrast to the ample resources available to researchers using fluorescent proteins of various wavelengths in rodents, fluorescent proteins available for researchers using porcine models remain under development. Our group [4] and Murakami et al [5] have generated green fluorescent protein (GFP)-transgenic porcine models. Sommer et al [6] produced transgenic pigs that expressed Stra8-enhanced yellow fluorescent protein.…”

Rapid Non-Invasive Genotyping of Reporter Transgenic Mammals

“…Recently, the generation of transgenic pigs with systemic expression of vital fluorophore markers has found widespread interest (311), e.g., for improving gene transfer technologies in pigs (3,5,79,11), for transplantation studies (4,10), for testing the functionality of multicistronic transgene constructs (2), and for visualizing epigenetic phenomena during spermatogenesis and fertilization (6). …”