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Skeletal Muscle Fiber-Specific Green Autofluorescence: Potential for Stem Cell Engraftment Artifacts

^a Center for Cell and Gene Therapy,
^b Department of Molecular and Cellular Biology, Integrated Microscopy Core, and
^c Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA

Key Words. Skeletal muscle • Autofluorescence • Stem cell plasticity

Margaret A. Goodell, Ph.D., Center for Cell and Gene Therapy and Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, BCM N1050, Houston, Texas 77030, USA. Telephone: 713-798-1269; Fax: 713-798-1230; e-mail: goodell{at}bcm.tmc.edu

	ABSTRACT

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Adult stem cell research has lately been plagued by controversy regarding the possibility that some adult stem cells can engraft into nonautochthonous tissues. While most reports have observed some level of engraftment, the prevalence has varied in some cases by two orders of magnitude, suggesting that major technical variations may underlie these differences, possibly outweighing the biological basis of the observations. Here we describe bright green autofluorescence in a specific subset of skeletal muscle fibers that strongly resembles emission from green fluorescent protein (GFP). Moreover, we show that oxidative muscle fibers exhibit this autofluorescence, likely due to flavin, associated with NADH dehydrogenase. Finally, we demonstrate that confocal microscopy, in conjunction with spectral scanning, can be used to distinguish between GFP and autofluorescence. We suggest this autofluorescence artifact may account for some of the discrepancies in this field, particularly those describing skeletal muscle engraftment.

	INTRODUCTION

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The use of green fluorescent protein (GFP), instead of ß-galactosidase as a marker in transgenic mice is becoming increasingly common. While GFP provides some advantages over histo- or immunohistochemical marker systems, particularly in terms of live imaging, some tissues have high levels of autofluorescence that can confound analysis. Such autofluorescence can be particularly problematic when using sensitive imaging techniques to look for rare events in a tissue. We became acutely aware of this while investigating potential engraftment of hematopoietic stem cells (HSCs) into skeletal muscle.

The concept of "transdifferentiation" of adult stem cells, particularly HSCs, into nonautochthonous cell types has received a great deal of interest recently due to the potential offered for cellular therapy [1]. Generation of skeletal muscle from HSCs was one of the first areas of interest, but reported efficiencies vary by two orders of magnitude from ~0.05% [2, 3], to ~3% [4–6]. Contribution of wild-type cells to dystrophic muscle could potentially be therapeutic in the range of 1%-10%. Considering the enormous disparities observed in engraftment levels, it is vital to identify the baseline engraftment potential of bone-marrow-derived cells into skeletal muscle in order to determine whether bone marrow transplantation may be useful for the treatment of skeletal muscle disorders in the near future.

Although engraftment levels vary from laboratory to laboratory, within a research group engraftment levels remain constant. This suggests that the differences observed in engraftment levels may be due to the techniques used to identify the transdifferentiation event. The presence of the Y chromosome [3], GFP expression [4–6], and nuclear-localized ß-galactosidase [2] expression have all been used to identify skeletal muscle fibers containing bone-marrow-derived cells. Thus far, experiments performed using bone marrow cells expressing GFP have resulted in the highest levels of muscle engraftment. It has been suggested that the lower engraftment levels observed in experiments using ß-galactosidase as a marker may be due to silencing of the lacZ gene. Here, while performing preliminary experiments to determine whether we could detect engraftment from GFP-positive cells in skeletal muscle, we observed a high level of fiber-type-specific green autofluorescence that could potentially be mistaken for GFP fluorescence.

	MATERIALS AND METHODS

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Tissue Isolation
C57Bl/6 and C57Bl/6-TgN (ACTßEGFP)1Osb mice (The Jackson Laboratory; Bar Harbor, ME; http://www.jax.com) were used for the experiments outlined in this paper. Mice used for examination of background fluorescence were 9 months of age, while mice used in muscle regeneration experiments and GFP mice were 6–12 weeks of age. Tissue fixation was performed via perfusion through the left ventricle with phosphate-buffered solution (PBS) followed by 1.5% paraformaldehyde (PFA)/0.5% glutaraldehyde. The tibialis anterior muscle was removed and snap frozen in liquid-nitrogen-chilled isopentane. Muscles were frozen sectioned at a thickness of 10 µm or 40 µm, mounted on slides, and visualized by microscopy.

Determination of Muscle Fiber Type
Serial sections of 10 µm each were made from unperfused unfixed tissue and were stained for myosin ATPase activity and NADH activity. NADH activity was determined by a 30-minute incubation at 37°C with 1.5 mM NADH (Sigma-Aldrich; St. Louis, MO; http://www.sigma-aldrich.com) and 1.5 mM Nitro blue tetrazolium (Sigma-Aldrich) in 0.2 M Tris (Sigma-Aldrich). Myosin ATPase staining was performed using the protocol of Guth and Samaha [7]. Briefly, frozen sections were air dried for 30 minutes followed by preincubation in alkaline and acidic solutions. Alkali preincubation was carried out for 15 minutes at pH 10.4 using 0.1 M 2-amino-2-methyl-1-propanol (Sigma-Aldrich) containing 18 mM CaCl₂, while acidic preincubation occurred at pH 4.0 in 0.1 M potassium acetate buffer for 10 minutes. Slides were rinsed with 0.1 M Tris-HCl and incubated for 30 minutes at 37°C in a buffer solution of 0.1 M 2-amino-2-methyl-1-propanol (pH 9.4) containing 18 mM CaCl₂ and 2.7 mM ATP (Sigma-Aldrich). ATPase activity was visualized by staining in 2% cobalt chloride (Sigma-Aldrich) followed by 1% ammonium sulfide (Sigma-Aldrich).

Myoblast Transplantation
The tibialis anterior (TA) muscles of C57Bl/6 mice were injured by a 20-µl injection of cardiotoxin (1 mg/ml, Sigma-Aldrich). Twenty-four hours later, 1 x 10⁵ muscle cells isolated by type II collagenase digestion (0.2%; Worthington Biochemical Corporation; Lakewood, NJ; http://www.worthington-biochem.com) [8] from a GFP mouse were injected into the injured TA. Two weeks following injury, mice were perfused with 1.5% PFA/0.5% glutaraldehyde, as described above. TA muscles were snap frozen, sectioned, and visualized by microscopy.

Microscopy
Routine microscopy was performed on a Zeiss Axioplan 2 using a fluorescein isothiocyanate (FITC) filter set with an excitation of BP 450–490 and an emission of BP 515–565. Digital images were taken with a Photometrics CoolSnap CF (Roper Scientific; Trenton, NJ; http://www.roperscientific.com) camera using Metavue (Nikon) imaging software. Confocal microscopy was done on a Zeiss LSM510 equipped with the Meta spectral scanning unit. Excitation was done at 488 nm with an HFT UV/488/543/633 dichroic mirror, while 11 nm emission bandpass windows were collected from 494–580 nm. All images were collected with a Plan NeoFluar 40 x 1.30 NA objective.

	RESULTS

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Skeletal Muscle Autofluorescence
Prior to embarking on experiments to examine the prevalence of hematopoietic stem cell derived engraftment into skeletal muscle, we wanted to ensure that GFP could be readily detected in mature muscle fibers from GFP mice, and that muscle fibers produced upon regeneration with myogenic stem cells from the labeled mice could be detected in a background of negative cells. Using the TA muscle, a small muscle on the lower hind leg of mice, we observed bright green fluorescent muscle fibers in wild-type mice that expressed enhanced GFP fluorescence under a chicken ß-actin promoter (GFP mice, Fig. 1A). To test GFP fluorescence in regenerating muscles, we used a standard regeneration assay, wherein myogenic stem cells derived from GFP mice were injected into injured muscles of normal GFP-negative mice. The injury was induced by injection of cardiotoxin, derived from cobra venom, which destroys mature muscle fibers, allowing robust regeneration from myogenic stem cells to occur [8]. Mononuclear muscle cell preparations that contained myogenic stem cells were prepared from GFP mice and injected in this assay, resulting in a subset of bright green fibers, as shown in Figure 1B. Note the variety of fiber sizes in this panel and the wide spacing between them, compared with the uninjured muscle (Fig. 1A), indicating the ongoing regeneration. However, the negative controls, involving uninjured, uninjected muscles of normal GFP-negative mice that were prepared under identical conditions also displayed a distinct subset of muscle fibers that were similarly bright green, with the clear appearance of GFP expression (Fig. 1C and Fig. 1D). These fibers were consistently of a smaller diameter than adjacent non-fluorescent fibers. Since regenerating fibers tend to be smaller, this autofluorescence could suggest correlation with regeneration, even though no regeneration should be occurring in these negative-control mice, and no GFP-positive cells are present. Even in the sections from GFP mice (Fig. 1A), one can observe a subset of fibers that are a brighter green than others, and that also have a smaller diameter. The brighter green of these fibers presumably reflects the addition of GFP and autofluorescence.

View larger version (112K): [in this window] [in a new window]   Figure 1. GFP fluorescence and autofluorescence in skeletal muscle. A) Section of TA muscle from normal GFP transgenic mice (chicken ß-actin promoter). No image editing was performed for any of these images. All sections shown in this figure were from animals perfused with 1.5% PFA/0.5% glutaraldehyde prior to excision of the muscle and frozen sectioning. Sections are 10 µm. Photograph was taken for 5 milliseconds. B) Section of regenerating TA muscle injected with satellite cells derived from a GFP transgenic mouse (above). Photograph taken for 5 milliseconds. The TA of a normal GFP-negative wild-type mouse was injured by injection with cardiotoxin followed after 24 hours with GFP+ satellite cells. Two weeks later, the animals were sacrificed, perfused and the TA frozen sectioned. C) Cross-section of TA from a normal GFP-negative C57Bl/6 mouse after perfusion and frozen sectioning, performed identically as for the images in A and B. Photograph exposed for 75 milliseconds. D) Longitudinal sections of TA as in C. Exposure time 75 milliseconds.

Regarding fixation conditions, we had initially followed protocols in the literature that suggested perfusion fixation in 1.5% PFA/0.5% glutaraldehyde [4]. Muscles fixed under such conditions are shown in Figure 1. Fixation of muscle tissue after sectioning rather than by perfusion resulted in an overall increase in nonspecific background autofluorescence, which abolished the ability to distinguish a specific subset of autofluorescent fibers (not shown). In unfixed tissue, the distinct autofluorescence in specific fibers was still readily detectable, but the fluorescence was generally more punctate and concentrated at the edges of the fibers (Fig. 2E), making it slightly more difficult to identify. Glutaraldehyde was the essential component, as perfusion fixation with PFA alone resulted in staining most similar to unfixed tissue (not shown). Since perfusion fixation enhances the ability to detect the autofluorescent fibers, variations in the effectiveness of perfusion resulted in some animals appearing to have a higher prevalence of autofluorescence.

View larger version (89K): [in this window] [in a new window]   Figure 2. Type IIa muscle fibers exhibit autofluorescence. A through D and E through G are 10 µm serial sections through nonfixed TA muscle from a normal GFP-negative C57Bl/6 mouse. A) Section of TA muscle demonstrating readily identifiable autofluorescent fibers in a standard fluorescein channel. B) Adjacent section stained for NADH hydroxylase activity. The dark blue fibers have the highest levels of NADH hydroxylase, showing that autofluorescent fibers are oxidative. C) and D) Characterization of fiber type. A section adjacent to A/B was stained for myosin ATPase activity after preincubation at pH 10.4 (C), with dark black staining being characteristic of type IIa fibers, or after incubation at pH 4.0 (D), with dark staining indicative of type I fibers. Since no type I fibers were present in this section, another area of the TA muscle that contained these was analyzed in E-G. E) Section of TA muscle with autofluorescent fibers. F) Adjacent section stained for myosin ATPase activity at pH 10.4 for type IIa fibers. G) Adjacent section stained for myosin ATPase activity at pH 4.0. The dark fibers in this section are type 1, revealing that these fibers are not autofluorescent. Exposure time for all fluorescent panels was 75 milliseconds.

Oxidative fibers can be subdivided into type IIa fast and type I slow twitch, which can be distinguished by the sensitivity of the myosin ATPase contained within these fibers to alkaline or acidic pH [10]. Preincubation of skeletal muscle sections at an alkaline pH followed by staining for myosin ATPase activity results in a heavy staining of type IIa fibers, intermediate staining of type IIb fibers, and no staining of type I fibers. In contrast, preincubation at an acidic pH results in ATPase activity in only type I fibers. When TA muscle sections were preincubated at pH 10.4, the darkly stained type IIa fibers correlated with the green fluorescent fibers (compare Fig. 2A with 2C, and 2E with 2F). When sections were incubated at pH 4.0 (Fig. 2D and 2G), the dark-stained type I fibers were not autofluorescent (no type I fibers are present in Fig. 2D; compare Fig. 2E with 2G).

Strategies for Distinguishing GFP from Autofluorescence
Because autofluorescence closely resembles GFP fluorescence, as shown in Figure 1, we attempted to develop a standard method to distinguish autofluorescence from true GFP fluorescence. Variable levels of fluorescence between controls and GFP-expressing tissues were monitored during microscopy by recording the exposure times used for each photo as noted in the figure legends (pictures were not manipulated with image-editing software). As long as we utilized positive and negative controls, imaged alongside the experimental samples, this method of separating autofluorescence from GFP fluorescence was acceptable in experiments that used GFP-marked satellite cell incorporation into regenerating muscle fibers because satellite cells contribute numerous nuclei to the regenerating muscle fibers, thereby resulting in high levels of GFP expression. In contrast, it has been shown that bone marrow contributes many fewer nuclei to a single regenerating muscle fiber [2, 3], rendering this method of distinguishing background fluorescence from GFP expression less reliable where contribution of true GFP-positive nuclei is rare.

Since all fluorophores have unique absorption and emission spectra, we investigated whether we could use confocal laser scanning microscopy (CLSM) to identify specific emission wavelengths with which to distinguish GFP fluorescence from autofluorescence. Our CLSM configuration excites a fluorophore using a 488-nm laser, and emissions are collected over a specified range in 11-nm increments. Thus, we scanned GFP⁺ and normal mouse TA muscle between 494 nm and 580 nm emission wavelengths. As shown in Figure 3A, the GFP fluorescence was brightest in a narrow range from 511 nm to 532 nm, whereas the autofluorescence appeared in this range, but also at higher wavelengths (Fig. 3B). This can be viewed graphically by measuring emission in a small area comprised of a single fiber and plotting the relative emission intensity versus wavelength, as shown in Figure 4. The overlay of the emission profiles of a bright GFP fiber and a bright autofluorescent fiber shows that both types of fluorescence are readily detected at around 520 nm, whereas the autofluorescence was much brighter above 560 nm. When this method was used to examine whether muscle fibers regenerated from GFP⁺ satellite cells (such as shown in Fig. 1B), we could readily distinguish GFP fluorescence from autofluorescence based on these typical profiles. These scans also demonstrate that judicious use of emission collection filters could at least reduce some background: a narrow bandpass filter optimized for GFP emission would reduce emissions coming from higher wavelengths derived from autofluorophores, whereas a longpass filter collecting a large range of wavelengths would be more prone to artifacts.

View larger version (77K): [in this window] [in a new window]   Figure 3. Scanning confocal microscopy of GFP+ and autofluorescent muscle. A) CLSM on GFP+ muscle was performed at the indicated wavelengths (numbers in white on each image). Sections from GFP-positive and negative mice were prepared as described for Figure 1. B) CLSM on GFP-negative cells. Note that the sensitivity of the detectors had to be increased to obtain sufficient signal, when scanning at such narrow wavelengths, on the GFP-negative tissue. This accounts for the somewhat grainy appearance in these unmanipulated images.

View larger version (18K): [in this window] [in a new window]   Figure 4. Graphical display of GFP fluorescence and autofluorescence intensity with wavelength. As indicated, the relative intensity of fluorescence from green fibers that were either GFP+ or autofluorescent is plotted against emission wavelength. The overlay shows that the minimum wavelength to detect autofluorescence is higher, as is the maximum range. Long-pass filters would, thus, allow substantially more autofluorescence to be detected than narrow band-pass filters.

	DISCUSSION

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Since bona fide GFP fluorescence is much brighter, religious use of positive and negative controls will allow the investigator to determine appropriate exposure times that will allow GFP fluorescence to be preferentially recorded. When total GFP contribution to a cell is low (particularly possible in large syncytial muscle fibers), this strategy may be insufficient. In this case, the emission spectra can be utilized.

On standard fluorescence microscopes, filters for GFP should be used that specifically exclude the highest autofluorescence wavelengths. As shown in Figure 3, a band-pass filter that passes wavelengths between 510 nm and 530 nm will allow the majority of GFP fluorescence to be detected while eliminating the large high wavelength autofluorescence peak. In conjunction, a longpass filter that enables detection of the higher autofluorescence wavelengths (~550 nm and above), will allow detection of autofluorescent fibers, allowing these to be disregarded in analysis.

To unequivocally distinguish low GFP contribution from autofluorescence, investigators could also use the latest generation of confocal microscopes, equipped with the capacity for spectral scanning. This method could, in principle, be applied to any of the new fluorochromes such as dsRed, yellow fluorescence protein (YFP), etc., where cellular autofluorescence may overlap in emission profiles. The available software is also capable of unmixing the fluorescent emission spectra from multiple fluorochromes. For example, if the spectra of the GFP-fluorescent as well as the autofluorescent signals are independently determined by scanning on control samples, the relative contributions of these two signals to fluorescence in any cell or fiber can be determined. This technique may be valuable not only for studying engraftment of stem cells, but also for modern cell biology experiments where fluorescently tagged intracellular proteins may be expressed at low levels and their intensities approach those of autofluorochromes.

What Accounts for Green Autofluorescence?
The phenomenon of green autofluorescence and the potential for confusion with GFP fluorescence has been noted previously [11]. There are three main autofluorophores that are prime candidates, namely NADH, lipofuscin, and flavin. Although fixation conditions change emission spectra to some degree, NADH has an emission peak between 440 nm and 470 nm, and is best excited with wavelengths in the UV range, so NADH is unlikely to be the culprit here. Lipofuscin is a poorly characterized substance containing lipids, carbohydrates, and proteins, thought to be more prevalent in older animals and in certain cell types, particularly in the brain. While this may be responsible for the autofluorescence observed in some reports [12–14], there is no evidence that lipofuscin is found in skeletal muscle. The most likely candidate here is flavin and flavin-containing proteins with an efficient absorption at 488 nm (thus excited by the blue light lasers used in flow cytometry and confocal microscopy) and a broad emission peak between 520 nm and 560 nm. Flavins, including flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are ubiquitous coenzymes involved in catabolism of pyruvate, fatty acids, and amino acids and, most importantly, in several enzymes of the electron transport pathway of mitochondria. FMN is, in fact, a prosthetic group of NADH dehydrogenase that catalyzes electron transfer from NADH to the next member of the electron transport chain. The type II oxidative fibers, as defined by the highest level of NADH dehydrogenase activity, showed the highest level of autofluorescence (Fig. 2), correlating with the presence of the flavoenzyme. Therefore, any cell type that is intensely using flavin-linked enzymes, particularly prevalent in mitochondrial respiration as in muscle, may exhibit similar green autofluorescence. Indeed, flavin-associated autofluorescence has been noted in many cell types including rat neurons and mouse macrophages [15]. We also note that, since type IIa fibers account for the autofluorescence, any treatment of the animals that would result in a relative increase in this fiber type would result in an increase in this apparent GFP fluorescence. Thus, conditions such as age, exercise, and muscle type that affect fiber composition would also change overall skeletal muscle fiber-type-specific autofluorescence.

The Use of GFP for Tracking Engraftment
With this in mind, it is worth considering the possibility that this distinct autofluorescence has been mistaken for GFP fluorescence in some reports, particularly when examining fluorescence in skeletal muscle [4–6]. During muscle regeneration, normally quiescent satellite cells become activated, divide, migrate, and fuse to generate mature muscle fibers, the fiber itself being a syncytium of hundreds of fused myoblasts. The nuclei of these myoblasts are centrally located shortly after fusion, but later migrate to the periphery of the muscle fiber where they are normally found. The cytoplasms of these fusing myoblasts are, thus, distributed within the regenerating fiber, resulting in diffusion of cytoplasmic proteins from a single myoblast within the muscle fiber. If only a small number of marked cells are involved in the fusion, as has been shown in early reports and confirmed by unpublished data from our laboratory [2, 3], a cytoplasmic marker protein would likely diffuse and become so diluted as to become undetectable. Therefore, at least in the muscle, nuclear markers such as Y-chromosome fluorescence in situ hybridization (FISH) or nuclear-localized lacZ (or nuclear-GFP) are likely to be more reliable, particularly for quantitative analysis of expression and engraftment. In order to study the discrepancy between muscle engraftment based on GFP versus lacZ, the most definitive experiment would be to cross the two mouse strains to create a donor with both markers. This would allow direct assessment of the correlation between these markers and also would reveal whether cytoplasmic GFP can be detected after diffusion throughout a syncitial muscle fiber.

Could this autofluorescence artifact also account for some of the other discrepancies in the field? Bone marrow engraftment into cardiac muscle has been reported, with a difference in reported prevalence of four orders of magnitude between studies [16–19], some of which have been based on GFP as a marker. In addition, frequencies of engraftment of bone-marrow-derived cells into the brain have varied according to the marker system used [14, 20, 21]. Since some of the corroborating studies also use the Y chromosome as a marker, autofluorescence cannot be the only explanation for these discrepancies, but bears consideration in interpreting these and other studies.

In summary, we have shown here that a specific subset of muscle fibers exhibits a strong autofluorescence that could easily be mistaken for bona fide GFP fluorescence. Certainly, the fluorescence from GFP is brighter and more intense. Thus, photographic exposure time can be a reliable indicator of autofluorescence, as long as GFP-negative and GFP-positive samples are imaged at the same time. In addition, judicious use of emission filters on standard epifluorescent microscopes, or confocal laser scanning microscopy, can be used to determine the identity of a fluorochrome in cases of doubt about the origin of green fluorescence. Where incorporation of GFP-positive cells into a tissue is expected to be rare, this method may be essential to distinguish autofluorescence from GFP positivity.

While GFP has been a revolutionary marker due to its ease of use, it has long been recognized that its fluorescence emission profile is close to that of some autofluorochromes and that this risks confounding interpretation in some types of experiments [11]. Here we exhibit one major drawback of GFP in some cell types. However, with careful use of controls, coupled with a minimalist approach to electronic processing of images both before and after photography, we believe GFP can continue to be an informative and powerful marker.

	ACKNOWLEDGMENT

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This work was funded by Muscular Dystrophy Association grants to M.A.G. and K.A.J. M.A.G. is a Scholar of the Leukemia and Lymphoma Society, and K.A.J. was a Fellow of the Leukemia and Lymphoma Society. We thank Michael Mancini of the Baylor College of Medicine and Department of Molecular and Cellular Biology Integrated Microscopy Core for suggestions.

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