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Infrared fluorescent protein 1.4 genetic labeling tracks engrafted cardiac progenitor cells in mouse ischemic hearts.

Chen L, Phillips MI, Miao HL, Zeng R, Qin G, Kim IM, Weintraub NL, Tang Y - PLoS ONE (2014)

Bottom Line: Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC.At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal.Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing, China; Department of Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America.

ABSTRACT
Stem cell therapy has a potential for regenerating damaged myocardium. However, a key obstacle to cell therapy's success is the loss of engrafted cells due to apoptosis or necrosis in the ischemic myocardium. While many strategies have been developed to improve engrafted cell survival, tools to evaluate cell efficacy within the body are limited. Traditional genetic labeling tools, such as GFP-like fluorescent proteins (eGFP, DsRed, mCherry), have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent these limitations, a near-infrared fluorescent mutant of the DrBphP bacteriophytochrome from Deinococcus radiodurans, IFP1.4, was developed for in vivo imaging, but it has yet to be used for in vivo stem/progenitor cell tracking. In this study, we incorporated IFP1.4 into mouse cardiac progenitor cells (CPCs) by a lentiviral vector. Live IFP1.4-labeled CPCs were imaged by their near-infrared fluorescence (NIRF) using an Odyssey scanner following overnight incubation with biliverdin. A significant linear correlation was observed between the amount of cells and NIRF signal intensity in in vitro studies. Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC. To assess efficacy of our model for engrafted cells in vivo, IFP1.4-labeled CPCs were intramyocardially injected into infarcted hearts. NIRF signals were collected at 1-day, 7-days, and 14-days post-injection using the Kodak in vivo multispectral imaging system. Strong NIRF signals from engrafted cells were imaged 1 day after injection. At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal. The data collected 2 weeks following transplantation showed an 88% decrease when compared to day 1. Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo.

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Assessment of signal consistence of IFP1.4 labeled CPC.(A) Correlation between CPC numbers and near infrared signals, IFP1.4-labeled CPC at 2.5×105, 1.25×105, 6.25×104, 3.125×104, and 1.57×104 were seeded into 24-well plate, and incubated with 25 µM biliverdin overnight, and then subjected to scanning on the Odyssey Infrared imager; (B) Assessment of near infrared signal intensity showed a robust linear correlation (R2 = 0.9927) between the cell number and near infrared signal; (C–D) Comparison of near infrared signal from CPC IFP1.4 at P6, P7, P8 and P9 with/without Biliverdin treatment.
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pone-0107841-g003: Assessment of signal consistence of IFP1.4 labeled CPC.(A) Correlation between CPC numbers and near infrared signals, IFP1.4-labeled CPC at 2.5×105, 1.25×105, 6.25×104, 3.125×104, and 1.57×104 were seeded into 24-well plate, and incubated with 25 µM biliverdin overnight, and then subjected to scanning on the Odyssey Infrared imager; (B) Assessment of near infrared signal intensity showed a robust linear correlation (R2 = 0.9927) between the cell number and near infrared signal; (C–D) Comparison of near infrared signal from CPC IFP1.4 at P6, P7, P8 and P9 with/without Biliverdin treatment.

Mentions: We have constructed a lentiviral vector containing IFP1.4 gene to stably express IFP1.4 protein in mouse CPC (Fig. 2A), in order to purify the IFP1.4-expressing CPC, we subjected them to puromycin selection at the concentration of 5, 7.5 and 10 ug/ml, as shown in Fig. 2B, selection by puromycin at the highest dose (10 µg/ml) gave high proportion of IFP1.4-positive cells with high level of fluorescent signals. Next, we incubated IFP1.4-positive cells (selection with 10 µg/ml puromycin) with biliverdin for extended period of time, ranging from 0–14 hrs; obvious IFP1.4 signals were observed after a 5-hour incubation when 25 µM biliverdin was applied. The strongest signal appears after cell exposure at 14 hrs (Fig. 2C–D), suggesting time dependent IFP1.4 signal. Finally, we investigate whether IFP1.4 labeling can be used for cell quantification accurately. First, we evaluated the correlation between near infrared signal and the amount of seeding cells. We observed a significant linear correlation existed between the amount of cells (x) and near infrared signal values (y). The square of the correlation coefficient (R2) was 0.9927 (Fig. 3A–B), which suggests that IFP1.4 molecular signal enables accurate cell quantification in vitro – an important feature for assessing cell proliferation. Persistence of IFP1.4 signal from labeled cells is important for us to determine whether this technology can be used for assaying in vivo stem cell survival, we evaluated the persistence of the IFP1.4 fluorescent signal of CPC for four passages (P6-P9) with time interval at 1 week intervals. As demonstrated in Fig. 3C–D, lentiviral mediated IFP1.4 gene labeling shows persistent signal at least three weeks without decay, suggesting that IFP1.4 molecular labeling enables real-time assessment of cell survival in vivo.


Infrared fluorescent protein 1.4 genetic labeling tracks engrafted cardiac progenitor cells in mouse ischemic hearts.

Chen L, Phillips MI, Miao HL, Zeng R, Qin G, Kim IM, Weintraub NL, Tang Y - PLoS ONE (2014)

Assessment of signal consistence of IFP1.4 labeled CPC.(A) Correlation between CPC numbers and near infrared signals, IFP1.4-labeled CPC at 2.5×105, 1.25×105, 6.25×104, 3.125×104, and 1.57×104 were seeded into 24-well plate, and incubated with 25 µM biliverdin overnight, and then subjected to scanning on the Odyssey Infrared imager; (B) Assessment of near infrared signal intensity showed a robust linear correlation (R2 = 0.9927) between the cell number and near infrared signal; (C–D) Comparison of near infrared signal from CPC IFP1.4 at P6, P7, P8 and P9 with/without Biliverdin treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214633&req=5

pone-0107841-g003: Assessment of signal consistence of IFP1.4 labeled CPC.(A) Correlation between CPC numbers and near infrared signals, IFP1.4-labeled CPC at 2.5×105, 1.25×105, 6.25×104, 3.125×104, and 1.57×104 were seeded into 24-well plate, and incubated with 25 µM biliverdin overnight, and then subjected to scanning on the Odyssey Infrared imager; (B) Assessment of near infrared signal intensity showed a robust linear correlation (R2 = 0.9927) between the cell number and near infrared signal; (C–D) Comparison of near infrared signal from CPC IFP1.4 at P6, P7, P8 and P9 with/without Biliverdin treatment.
Mentions: We have constructed a lentiviral vector containing IFP1.4 gene to stably express IFP1.4 protein in mouse CPC (Fig. 2A), in order to purify the IFP1.4-expressing CPC, we subjected them to puromycin selection at the concentration of 5, 7.5 and 10 ug/ml, as shown in Fig. 2B, selection by puromycin at the highest dose (10 µg/ml) gave high proportion of IFP1.4-positive cells with high level of fluorescent signals. Next, we incubated IFP1.4-positive cells (selection with 10 µg/ml puromycin) with biliverdin for extended period of time, ranging from 0–14 hrs; obvious IFP1.4 signals were observed after a 5-hour incubation when 25 µM biliverdin was applied. The strongest signal appears after cell exposure at 14 hrs (Fig. 2C–D), suggesting time dependent IFP1.4 signal. Finally, we investigate whether IFP1.4 labeling can be used for cell quantification accurately. First, we evaluated the correlation between near infrared signal and the amount of seeding cells. We observed a significant linear correlation existed between the amount of cells (x) and near infrared signal values (y). The square of the correlation coefficient (R2) was 0.9927 (Fig. 3A–B), which suggests that IFP1.4 molecular signal enables accurate cell quantification in vitro – an important feature for assessing cell proliferation. Persistence of IFP1.4 signal from labeled cells is important for us to determine whether this technology can be used for assaying in vivo stem cell survival, we evaluated the persistence of the IFP1.4 fluorescent signal of CPC for four passages (P6-P9) with time interval at 1 week intervals. As demonstrated in Fig. 3C–D, lentiviral mediated IFP1.4 gene labeling shows persistent signal at least three weeks without decay, suggesting that IFP1.4 molecular labeling enables real-time assessment of cell survival in vivo.

Bottom Line: Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC.At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal.Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing, China; Department of Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America.

ABSTRACT
Stem cell therapy has a potential for regenerating damaged myocardium. However, a key obstacle to cell therapy's success is the loss of engrafted cells due to apoptosis or necrosis in the ischemic myocardium. While many strategies have been developed to improve engrafted cell survival, tools to evaluate cell efficacy within the body are limited. Traditional genetic labeling tools, such as GFP-like fluorescent proteins (eGFP, DsRed, mCherry), have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent these limitations, a near-infrared fluorescent mutant of the DrBphP bacteriophytochrome from Deinococcus radiodurans, IFP1.4, was developed for in vivo imaging, but it has yet to be used for in vivo stem/progenitor cell tracking. In this study, we incorporated IFP1.4 into mouse cardiac progenitor cells (CPCs) by a lentiviral vector. Live IFP1.4-labeled CPCs were imaged by their near-infrared fluorescence (NIRF) using an Odyssey scanner following overnight incubation with biliverdin. A significant linear correlation was observed between the amount of cells and NIRF signal intensity in in vitro studies. Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC. To assess efficacy of our model for engrafted cells in vivo, IFP1.4-labeled CPCs were intramyocardially injected into infarcted hearts. NIRF signals were collected at 1-day, 7-days, and 14-days post-injection using the Kodak in vivo multispectral imaging system. Strong NIRF signals from engrafted cells were imaged 1 day after injection. At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal. The data collected 2 weeks following transplantation showed an 88% decrease when compared to day 1. Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo.

Show MeSH
Related in: MedlinePlus