Effect of dual-optical pulses with temporal energy distribution on laser ablation performance in in vivo zebrafish model.

A Q-switched laser system has been used in a single-pulse mode for skin melasma treatments because of instant heat deposition in the target. Despite the efficient ablation of the melanophores in the skin, the single, high-fluence pulse often causes undesirable damage to the surrounding tissue, leading to high recurrence rates. This study aims to investigate the feasibility of dual-optical pulses with a temporal energy distribution on the melasma treatment in in vivo zebrafish models in comparison to that of the single optical pulse. Based on the optical detection, the dual-optical pulses had a temporal energy distribution ratio of 4:1 and an interval of 61 µs between the two consecutive pulses. According to the histological analysis, the dual pulses removed melanophores and induced a few apoptotic nuclei with minimal recurrence. This study demonstrated that the feasibility of dual-optical pulses (energy ratio = 4:1) could enhance the laser ablation performance in vivo.

Feeder cell-dependent primary culture of single blastula-derived embryonic cell lines from marine medaka (Oryzias dancena).

Fish embryonic stem cells (ESCs) are derived from blastomeres that have been cultured from blastula embryos. The most widely used method for derivation of fish ESCs is the culture of blastomeres that have been isolated from approximately 10 blastula embryos under feeder-free conditions. However, this method leads to intercellular genetic heterogeneity among the cultured cells, which is a major obstacle to the development of stable ESC culture conditions. In this study, to establish ESC lines with intercellular genetic homogeneity at the early stage of culture, we attempted to derive embryonic cell lines from single blastula-derived blastomeres of marine medaka (Oryzias dancena) in a feeder cell culture system. Using basic fibroblast growth factor-expressing feeder cells during primary culture, we successfully established 22 single blastula-derived embryonic cell lines that could be subcultured more than 20 times. In contrast, we were unable to efficiently derive cell lines using wild-type feeder cells and under feeder-free conditions. The established cell lines exhibited ESC-like cell characteristics in terms of alkaline phosphatase activity, pluripotency-related gene expression, and embryoid body formation. The results of this study will contribute to the development of methods for derivation of fish ESCs.

Improved conditions of a whole testis organ culture system in terms of spermatogonial proliferation levels in marine medaka (Oryzias dancena).

In vitro spermatogenesis can be performed for marine medaka (Oryzias dancena) via whole testis organ cultures, but spermatogenesis could only be maintained during the early phase of culturing, suggesting that the culture conditions can be further optimized. To improve the culture conditions, we examined the effects of culture temperature, basal media, and medium supplements on spermatogonial proliferation levels during whole testis organ culturing by BrdU incorporation assays. Our results show that a 30°C culture temperature negatively affected spermatogonial proliferation compared to 26°C and 28°C and that the use of Dulbecco's Modified Eagle Medium and Minimum Essential Medium α (α-MEM) was more effective for spermatogonial proliferation than the use of Leibovitz's L-15 Medium (L15). When fetal bovine serum (FBS) was replaced with KnockOut Serum Replacement (KSR), a significantly positive effect was observed for the maintenance of spermatogonial proliferation. However, supplementation of the medium with 17α, 20ß-dihydroxy-4-pregnen-3-one did not show any significant effect. Gene expression analyses of four genes, including Nanos2, SCP3, AMH, and StAR, indicated that the optimized culture conditions consisting of α-MEM and KSR had the most positive influence on the maintenance of spermatogonial proliferation levels in whole testis organ cultures compared to the original culture conditions consisting of L15 and FBS by maintaining the function of Sertoli and Leydig cells. The results from this study will provide useful information for the study of in vitro spermatogenesis in fish.

Enhanced Enrichment of Medaka Ovarian Germline Stem Cells by a Combination of Density Gradient Centrifugation and Differential Plating.

Fish ovarian germline stem cells (OGSCs) have great potential in various biological fields due to their ability to generate large numbers of mature eggs. Therefore, selective enrichment of OGSCs is a prerequisite for successful applications. To determine the optimal conditions for the enrichment of OGSCs from Japanese medaka (Oryzias latipes), we evaluated the effects of Percoll density gradient centrifugation (PDGC), differential plating (DP), and a combination of both methods. Based on cell morphology and gene expression of germ cell-specific Vasa and OGSC-specific Nanos2, we demonstrated that of seven density fractions obtained following PDGC, the 30-35% density fraction contained the highest proportion of OGSCs, and that Matrigel was the most effective biomolecule for the enrichment of Oryzias latipes OGSCs by DP in comparison to laminin, fibronectin, gelatin, and poly-l-lysine. Furthermore, we confirmed that PDGC and DP in combination significantly enhanced the efficiency of OGSC enrichment. The enriched cells were able to localize in the gonadal region at a higher efficiency compared to non-enriched ovarian cells when transplanted into the developing larvae. Our approach provides an efficient way to enrich OGSCs without using OGSC-specific surface markers or transgenic strains expressing OGSC-specific reporter proteins.

Gene delivery into Siberian sturgeon cell lines by commercial transfection reagents.

The optimal transfection conditions for efficient transgene delivery into a specific cell type should be empirically determined, particularly in cases involving unusual cell types. We compared the conditions for effective introduction of transgenes into Siberian sturgeon (Acipenser baerii) cell lines by evaluating the cytotoxicity and transfection efficiency of three commercially available transfection reagents: Lipofectamine 2000, X-tremeGENE HP DNA Transfection Reagent, and GeneJuice Transfection Reagent. Plasmid vectors containing the gene encoding enhanced green fluorescent protein were mixed with each of the transfection reagents using reagent-to-plasmid ratios of 1:1, 2:1, and 4:1. Then, the complexes were used to transfect three Siberian sturgeon cell lines derived from the heart, head kidney, and gonad. Cytotoxicity and transfection efficiency were measured via flow cytometry after propidium iodide staining. No significant cytotoxicity was observed at the optimal treatment conditions in all cases, with the exception of Lipofectamine 2000-treated gonad-derived cells. Although the transfection efficiencies in A. baerii cells were generally low, X-tremeGENE HP DNA Transfection Reagent showed the highest transfection efficiency at ratios of 2:1 or 4:1, depending on the cell type. Hence, X-tremeGENE HP DNA Transfection Reagent can be used to effectively transfer foreign genes into three A. baerii cell lines.

Enhanced Adhesion of Fish Ovarian Germline Stem Cells on Solid Surfaces by Mussel-Inspired Polymer Coating.

Development of advanced cell culture methods has gained increasing attention because it allows for efficient genetic engineering and precise regulation of animal reproduction on a cellular basis. Numerous studies have attempted to develop an advanced cell culture method. Previous studies have altered cell culture media and pretreated culture plates with functional molecules. Among them, a mussel-inspired polymer coating has been extensively utilized owing to its wide applicability. For instance, adhesion of human embryonic stem cells and neuronal cells on solid surfaces has been improved. Despite the excellent capability of the mussel-inspired polymer coating, most studies have primarily focused on mammalian cells. However, the efficacy of these coatings on the adhesion of other cell lines is yet unclear. This study aimed to assess the potential of the mussel-inspired polymer coating in the regulation of the adhesion of fish ovarian germline stem cells on solid surfaces. Solid surfaces were coated by polydopamine and poly-L-lysine, and the effect of the coatings on cellular behaviors was investigated.

Non-lethal method for the preparation of metaphase spreads using cultured mantle tissue from live adult abalone.

Metaphase spread preparation in adult abalone has not been successful, which has restricted the applications of karyotyping-based technologies. Here, we present a non-lethal method to enable preparation of metaphase spreads from live adult abalone using a tissue culture method. Mantle tissue fragments from live adult abalone were cultured in vitro and the cultured cells were used for metaphase spread preparation. To retrieve a sufficient number of proliferating cells required for metaphase spread preparation, at least 14 days of culture was required, and culturing the marginal zone of mantle was more optimal than culturing other areas. Additionally, it was shown that simple medium consisting of basal medium, fetal bovine serum and antibiotics could stimulate cellular proliferation followed by metaphase spread preparation.

Gene expression profiles of human subcutaneous and visceral adipose-derived stem cells.

Subcutaneous and visceral adipose tissues show a different risk effect on metabolic disorders because they have distinct cellular properties. We isolated stem cells from the separate human adipose tissues to investigate that subcutaneous and visceral fat depots have metabolic differences. Adipose-derived stem cells (ASCs) were characterized by immunophenotype and differentiation potentials into adipogenic, osteogenic, and chondrogenic lineages. Although subcutaneous and visceral ASCs (S-ASC and V-ASC) express same surface markers (CD31- , CD34- , CD45- , CD73+ , CD90+ , and CD105+ ) and have differentiation potentials, S-ASCs had higher capacity to proliferate and to differentiate into adipogenic lineage than V-ASCs. Next, we identified that S-ASC and V-ASC were genetically distinct based on microarray analysis. Among a total of 810 genes detected in ASCs of both depots, the differentially expressed genes were involved in energy and lipid metabolism. These data show the existence of the intrinsic difference between S-ASC and V-ASC and suggest the differences of anatomically separated adipose tissue. On the basis of the differentially expressed gene profiles between S-ASC and V-ASC, we suggested significant evidence that adipose tissues originating from different anatomic regions are distinguished at the level of the undifferentiated stem cells such as mature adipocytes. V-ASCs had the upregulated clusters of genes related to lipid biosynthesis and metabolism. By contrast, S-ASCs highly expressed genes involved in DNA-dependent transcription, contributing to proliferation. We provide further insights for ASCs with the different origins to understand fat accumulation and distribution and a possibility of ASCs as a therapeutic target against metabolic disorders or cancer.

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena).

This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10 % DMSO, 20 % FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.

Identification of embryonic stem cell activities in an embryonic cell line derived from marine medaka (Oryzias dancena).

This study was conducted to identify embryonic stem cell (ESC) activities of a long-term cultured embryonic cell line previously derived from blastula-stage Oryzias dancena embryos. Five sub-cell lines were established from the embryonic cell line via clonal expansion of single cells. ESC activities, including clonogenicity, alkaline phosphatase (AP) activity, and differentiation capacity, were examined in the five sub-cell lines. We observed both clonogenicity and AP activity in all five sub-cell lines, but the proportion of cells that exhibited both properties was significantly different among them. Even though we detected different formation rates and sizes of embryoid body (EB) among these cells, all lines were stably able to form EBs and further induction for differentiation showed their capability to differentiate into other cell types in a spontaneous manner. From this study, we determined that the embryonic cell lines examined possessed heterogeneous ESC activities and can be utilized as a marine model system for fish ESC-based research.

Development of a chemically defined in vitro culture system to effectively stimulate the proliferation of adult human dermal fibroblasts.

Despite the fact that dermal fibroblasts are a practical model for research related to cell physiology and cell therapy, an in vitro culture system excluding serum, which complicates standardization and specificity and induces variability and unwanted effects, does not exist. We tried to establish a CDCS that supports effective proliferation of aHDFs. KDMEM supplemented with 5% (v/v) KSR, 12 ng/ml bFGF, 5 ng/ml EGF and 1 µg/ml hydrocortisone supported sufficient proliferation of aHDFs for 1 week. However, aHDF proliferation was decreased greatly after subculture. This problem could be overcome by culturing aHDFs in CDCM in culture plates coated with 10 µg/ml FN. Long-term culture of aHDFs was achieved using CDCM and FN-coated culture plates for 7 weeks. The optimized CDCS increased the proliferation of aHDFs significantly, without any increase in the senescence rate or alteration in morphology of aHDFs, despite long-term culture. In conclusion, we established a CDCS that improved proliferation of aHDFs while inhibiting cellular senescence. The CDCS will contribute to advances in various future research related to clinical skin regeneration.

The co-injection of somatic cells with embryonic stem cells affects teratoma formation and the properties of teratoma-derived stem cell-like cells.

The aim of this study was to assess the biological reactions triggered by stem cell transplantation related to phenotypic alteration, host-to-cell response, chromosomal stability, transcriptional alteration, and stem cell-like cell re-expansion. B6CBAF1 mouse embryonic stem cells (ESCs) were injected subcutaneously into homologous or heterologous (B6D2F1) recipients, and heterologous injections were performed with or without co-injection of B6D2F1 fetal fibroblasts. All homologous injections resulted in teratoma formation, whereas a sharp decrease in formation was detected after heterologous injection (100 vs. 14%; p<0.05). The co-injection of somatic cells in heterologous injections enhanced teratoma formation significantly (14 vs. 75%; p<0.05). Next, ESC-like cell colonies with the same genotype as parental ESCs were formed by culturing teratoma-dissociated cells. Compared with parental ESCs, teratoma-derived ESC-like cells exhibited significantly increased aneuploidy, regardless of homologous or heterologous injections. Repopulation of the parental ESCs was the main factor that induced chromosomal instability, whereas the co-injection of somatic cells did not restore chromosomal normality. Different genes were expressed in the parental ESCs and teratoma-derived ESC-like cells; the difference was larger with parental vs. heterologous than parental vs. homologous co-injections. The co-injection of somatic cells decreased this difference further. In conclusion, the host-to-cell interactions triggered by ESC transplantation could be modulated by co-injection with somatic cells. A mouse model using homologous or heterologous transplantation of stem cells could help monitor cell adaptability and gene expression after injection.

Establishment condition and characterization of heart-derived cell culture in Siberian sturgeon (Acipenser baerii).

This study was conducted to establish the efficient condition for stable derivation of heart-derived cell culture in Siberian sturgeon (Acipenser baerii). Three factors including isolation methods, cell densities in initial seeding, and basal media were evaluated for the derivation of heart-derived cell culture. As the results, enzymatic isolation was more efficient than mechanical isolation in both cell retrieval and further culture. Total 48 trials of culture employing low and middle cell densities of less than 5.5 × 10(4) cells/cm(2) in initial seeding did not induce cell survivals (0%, 0/48), but the trials in high cell density of more than 5.5 × 10(5) cells/cm(2) could induce cell survival and primary cell attachment on the plate (88.9%, 24 in 27 trials). When all initially attached cell populations were continuously cultured in two different media, only five cell populations that were enzymatically isolated and cultured under Leibovitz's L-15 medium could grow up to more than 40th subculture. Each cell population was stably cultured according to its own growth rate and all showed normal diploid DNA contents. Two morphologically different cell types that has an elongated shape or a round shape were identified in culture, which was subsequently identified that two cell types are considered as a fibroblast (an elongated shape) and a vascular endothelial cell (a round shape) on the basis of the results of gene and protein expression analysis. Additionally, the sufficient number of viable cells could be successfully retrieved after freezing and thawing from all five cell populations suggesting the feasibility of long-term cryopreservation of the cells. The data and cells obtained from this study will contribute to development of in vitro model for basic biological studies using sturgeon species.

Somatic cell transformation into stem cell-like cells induced by different microenvironments.

Development of induced pluripotent stem cell (iPSC) technology introduced a novel way to derive pluripotent stem cells, but the genetic manipulation required to generate iPSCs may lead to uncontrolled tumorigenesis of the established cells and thus limit clinical feasibility of the technology. Numerous attempts have been made to date, and alternative reprogramming of somatic cells to reactivate cellular plasticity after differentiation has been suggested. As a result, it had become clear that cell-to-cell interactions and specific acellular environments can be utilized for somatic cell reprogramming. In our previous studies, embryonic stem cell (ESC)-like cells could be derived from transforming ovarian cells and fetal fibroblasts by cell-to-cell interaction or specific cell-mediated microenvironmental factor(s). This cellular event was induced without undertaking genetic manipulation of progenitor cells. Several differences were found between the cellular properties of niche-induced, ESC-like cells and those of genetically manipulated iPSCs and the referenced ESCs. Thus, we provided evidence that terminally differentiated somatic cells either acquire pluripotency-like activity or possess cellular and genetic plasticity under a specific microenvironment and/or cell-to-cell interaction. In this minireview, we discuss derivation of stem cell-like cells under specific microenvironmental conditions in terms of technical perspectives and limitations.

Development of a serum-free defined system employing growth factors for preantral follicle culture.

This study was conducted to evaluate if mouse preantral follicles can yield developmentally competent oocytes following culture in serum-free, defined medium. Donor follicles were obtained from 14-day-old B6CBAF1 mice, and cultured in α-MEM-Glutamax medium. The replacement of fetal bovine serum with knockout serum replacement (KSR) did not significantly reduce follicle growth or oocyte maturation in vitro, although it significantly reduced the development of oocytes after activation. Regardless of the replacement medium, follicle growth was not influenced by the addition of leukemia inhibitory factor (LIF). The addition of 100 ng/ml stem cell factor (SCF) to the KSR-supplemented serum-free medium significantly stimulated follicle development, which further improved blastocyst formation after oocyte activation. On Day 3 of culture, a significant increase in Bmp7 expression was detected in the SCF-containing medium compared with the serum-containing medium, whereas Gdf9 and Amh were increased in the serum-containing medium. A significant increase in estradiol production was detected under serum-free conditions, but minimal progesterone secretion was detected throughout the culture period. In conclusion, serum-free media can be used to optimize ovarian follicle cultures, and the addition of SCF is beneficial for deriving developmentally competent oocytes through follicle culture.

Effects of combined antioxidant supplementation on human sperm motility and morphology during sperm manipulation in vitro.

OBJECTIVE: To evaluate the effect of antioxidant combinations on human sperm motility and morphologic normality during preparation and in vitro incubation to improve sperm quality for assisted reproductive technology (ART). DESIGN: Prospective study. SETTING: Laboratory. PATIENT(S): Six fertile males, 21 to 25 years of age, after 3 days of sexual abstinence. INTERVENTION(S): Sperm retrieved from patients with normal fertility prepared and incubated in vitro in medium supplemented with taurine, cysteine, and/or glutathione. MAIN OUTCOME MEASURE(S): Motility indices, including motility (MOT), average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), and morphologic normality. RESULT(S): Taurine supplementation during sperm preparation had no significant effects on motility or morphologic normality. However, when sperm was incubated for 24 hours under taurine-supplemented conditions, the rates of reduction in MOT, VAP, VCL, and VSL were statistically significantly decreased compared with taurine-free conditions. Morphologic normality was maintained, regardless of taurine treatment. The optimal taurine concentration for improving sperm motility and morphologic normality during preparation and in vitro incubation was 1 mM. Subsequently, combined treatment with 1 mM taurine, 1 mM cysteine, and 1 mM glutathione induced statistically significant synergistic effects on MOT, VAP, VCL, and VSL without any alteration of morphologic normality. CONCLUSION(S): Combined treatment with taurine, cysteine, and glutathione statistically significantly ameliorated the reduction in sperm motility during in vitro manipulation of human sperm.

Transformation of somatic cells into stem cell-like cells under a stromal niche.

To study the genomic plasticity of somatic cells without ectopic genetic manipulation, we cultured mouse fibroblasts with ovarian cells, embryonic fibroblasts of different strains, and parthenogenetic embryonic stem cells (ESCs). Of 41 trials, cell aggregation resembling nascent ESC colony from inner cell mass was detected in 9 cases (22%), and 6 cases (67%) yielded fibroblast-derived colonies with ESC morphology. Cells used in coculture provided the critical (P=0.0061) inducing factor for the aggregation. These colony-forming fibroblasts (CFFs) showed similar characteristics to those in ESCs and induced pluripotent stem cells (iPSCs), including pluripotency gene expression, in vitro differentiation, and teratoma formation. Furthermore, CFFs produced somatic chimera, although none showed germline chimerism. CFFs had a tetraploid-like karyotype, and their imprinting patterns differed from parthenogenetic ESCs, thereby confirming their nongermline transmissibility. We observed dysregulation of cell cycle-related proteins, as well as both homologous and heterologous recombination of genomic single-nucleotide polymorphisms in CFFs. Our observations provide information on somatic cell plasticity, resulting in stemness or tumorigenesis, regardless of colony-forming cell progenitors in the fibroblast population. The plasticity of somatic genomes under environmental influences, as well as acquisition of pluripotency by cell fusion, is also implicated.

Long-term maintenance of mouse embryonic stem cell pluripotency by manipulating integrin signaling within 3D scaffolds without active Stat3.

We engineered an acellular biomimetic microenvironment to regulate stem cell fate and applied it to maintain mouse embryonic stem (ES) cell self-renewal. In the 3D environment formed using hydrogel scaffolds in which specific integrin ligation was provided, Stat3 activation by exogenous leukemia inhibitory factor (LIF) no longer acted as a limiting factor for stem cell self-renewal. Instead, simultaneous stimulation of integrins α(5)ß(1), α(v)ß(5), α(6)ß(1) and α(9)ß(1) within the 3D scaffold greatly increased Akt1 and Smad 1/5/8 activation, which resulted in prolonged self-renewal of the ES cells. The ES cells exposed to the combined stimulation of the integrins for 4 wk in LIF-free 3D scaffolds maintained the spherical morphology of cell colonies without losing any activity of pluripotency. In conclusion, cell niche-specific integrin signaling within the 3D environment supported mouse ES cell self-renewal, and the resulting integrin signaling replaced Stat3 with Akt1 and Smad 1/5/8 as critical signals for mouse ES cell self-renewal.

Molecular cloning and characterization of three cDNAs encoding allatostatin-like neurosecretory peptides from Pandalopsis japonica.

Three cDNAs encoding allatostatin-like peptides (two myoinhibitory peptides; Paj-MIPI and Paj-MIPII, and one C-type AST; Paj-ASTC) were identified from Pandalopsis japonica through a combination of bioinformatic analysis and PCR-based gene cloning strategy. Paj-MIPI and Paj-MIPII encoded proteins with 189 and 117 amino acid residues, respectively, and a total of 10 mature peptides are putatively produced from the two MIP cDNAs (seven from Paj-MIPI and three from Paj-MIPII). Among the MIPs from various arthropods, their size and organization varied and it was unable to establish the monophyletic evolutionary relationship, which is mainly due to difference in the number and location of the mature peptide W(X(6))W motif of each MIP gene. Based on the sequence similarity of six residues flanked by two conserved tryptophan (W) residues, crustacean MIPs could be further classified into at least four groups. Paj-ASTC cDNA (648bp) encoded a protein with 143 amino acid residues. The prepropeptide of Paj-ASTC showed conserved C-type AST characteristics including a signal sequence, two dibasic cleavage sites, and a mature peptide sequence with two cysteine residues at the 7th and 14th positions, creating a disulfide bridge. Based on the sequence similarity in the mature peptides, the ASTCs in arthropods could be further classified into two subgroups, AVSCF-ASTC and PISCF-ASTC. Phylogenetic and sequence similarity analysis showed that Paj-ASTC belonged to the PISCF-ASTC subgroup. Expression studies revealed that AST-like peptides from P. japonica were mainly expressed in neuronal tissues, and the expression of Paj-ASTC was also detected in the intestine. Eyestalk ablation (ESA) altered the mRNA expression levels of both Paj-MIPs and Paj-ASTC, suggesting that factors from the sinus gland/X organ complex had a transient effect on AST-like peptide transcription. Correlation analysis of three allatostatin-like peptides revealed a strong positive correlation in brain tissues, suggesting that transcriptional regulation of three allatostatin-like peptides from P. japonica is influenced by the similar physiological condition.

Stem cell engineering: limitation, alternatives, and insight.

The 21st century will see improvements in the quality of human life. The development of new therapeutic technologies will prevent prevalent diseases and enable recovery from currently incurable diseases. The development of cell and tissue replacement therapies using stem cells and their progenitors will accelerate the development of causative treatments. The effort expended thus far in developing cell therapies has revealed many technical limitations. Thus, we must explore conceptual changes in the feasibility of stem cell therapy. This paper introduces the current limitations to stem cell engineering and ways to overcome these limitations, which will provide new insight into their clinical application.