Ulrik Frandsen

Teratoma Formation by Human Embryonic Stem Cells Is Site Dependent and Enhanced by the Presence of Matrigel

When implanted into immunodeficient mice, human embryonic stem cells (hESCs) give rise to teratoma, tumor-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data are available regarding the effects of implantation site and the methods employed for implantation on the success rate of teratoma formation. In this study, the rate of teratoma formation in immunodeficient mice was site dependent: subcutaneous (25-100%), intratesticular (60%), intramuscular (12.5%), and under the kidney capsule (100%). Co-injecting the hESCs with Matrigel increased subcutaneous teratoma formation efficiency from 25-40% to 80-100%. We did not observe site-specific differences in the teratoma composition at the histological level. However, subcutaneous teratomas were quite distinct, easy to remove, and caused minimal discomfort to the mice. Also, subcutaneous teratomas displayed larger proportion of solid tissues as opposed to cyst formation that dominated the teratomas formed at the other sites. Interestingly, a chromosomally abnormal hESCs with trisomy 20 formed teratomas where the ratio of differentiated to undifferentiated tissues was significantly decreased suggesting defective pluripotency of the cells. In conclusion, subcutaneous implantation of hESCs in presence of Matrigel appears to be the most efficient, reproducible, and the easiest approach for teratoma formation by hESCs. Also, teratoma formation can be employed to study the development defects exhibited by the chromosomally abnormal hESC lines.

Proliferation of myogenic stem cells in human skeletal muscle in response to low-load resistance training with blood flow restriction

In the last decade muscle training performed using a combination of low external loads and partial restriction of blood flow to the exercising limb has gained increasing interest, since it leads to significant gains in muscle strength and muscle mass. The cellular mechanisms responsible for the muscular adaptations induced by this training paradigm are not fully understood. This study shows that 3 weeks of high‐frequency, low‐intensity muscle exercise with partial blood flow restriction induces increases in maximal muscle strength accompanied by highly marked gains in muscle fibre size. Furthermore, the results indicate that these muscular adaptations rely on a considerable upregulation in myogenic satellite cells number, resulting in nuclear addition to the exercised myofibres. The results contribute to a better understanding of the physiological mechanisms underlying the gain in muscle strength and muscle mass observed with blood flow restricted low‐intensity resistance exercise.

Aging affects the transcriptional regulation of human skeletal muscle disuse atrophy.

Important insights concerning the molecular basis of skeletal muscle disuse-atrophy and aging related muscle loss have been obtained in cell culture and animal models, but these regulatory signaling pathways have not previously been studied in aging human muscle. In the present study, muscle atrophy was induced by immobilization in healthy old and young individuals to study the time-course and transcriptional factors underlying human skeletal muscle atrophy. The results reveal that irrespectively of age, mRNA expression levels of MuRF-1 and Atrogin-1 increased in the very initial phase (2–4 days) of human disuse-muscle atrophy along with a marked reduction in PGC-1α and PGC-1β (1–4 days) and a ∼10% decrease in myofiber size (4 days). Further, an age-specific decrease in Akt and S6 phosphorylation was observed in young muscle within the first days (1–4 days) of immobilization. In contrast, Akt phosphorylation was unchanged in old muscle after 2 days and increased after 4 days of immobilization. Further, an age-specific down-regulation of MuRF-1 and Atrogin-1 expression levels was observed following 2 weeks of immobilization, along with a slowing atrophy response in aged skeletal muscle. Neither the immediate loss of muscle mass, nor the subsequent age-differentiated signaling responses could be explained by changes in inflammatory mediators, apoptosis markers or autophagy indicators. Collectively, these findings indicate that the time-course and regulation of human skeletal muscle atrophy is age dependent, leading to an attenuated loss in aging skeletal muscle when exposed to longer periods of immobility-induced disuse.

Ageing is associated with diminished muscle re-growth and myogenic precursor cell expansion early after immobility-induced atrophy in human skeletal muscle

•  Elderly individuals require a prolonged recovery phase in order to return to initial muscle mass levels following short‐term immobilisation. •  The cellular mechanisms responsible for the attenuated re‐growth and associated molecular signalling processes in ageing human skeletal muscle are not fully understood. •  The main study finding was the observation of a less marked muscle mass recovery after immobilisation in elderly compared to young individuals that was paralleled by an elevation in myogenic precursor cell content in young individuals only, whereas the elderly failed to demonstrate any change in myogenic precursor cells. •  No age‐related differences were observed in the expression of major myogenic regulating factors known to promote skeletal muscle hypertrophy or satellite cell proliferation (IGF‐1Ea, MGF, MyoD1, myogenin, HGF gene products). •  In contrast, the expression of myostatin demonstrated a more pronounced up‐regulation following immobilisation along with an attenuated down‐regulation in response to reloading in older compared to young individuals, which may have contributed to the present lack of satellite cell proliferation in ageing muscle.

Aging impairs the recovery in mechanical muscle function following 4 days of disuse.

As aged individuals are frequently exposed to short-term disuse caused by disease or musculoskeletal injury, it is important to understand how short-term disuse and subsequent retraining affect lower limb mechanical muscle function. The purpose of the present study was, therefore, to investigate the effect of 4 days of lower limb disuse followed by 7 days of active recovery on mechanical muscle function of the knee extensors in young (24.3±0.9 years, n=11) and old (67.2±1.0 years, n=11) recreationally active healthy males. Slow and moderate dynamic muscle strength were assessed using isokinetic dynamometry (60 and 180° s(-1), respectively) along with isometric muscle strength and rapid muscle force capacity examined as contractile rate of force development (RFD), Impulse, and relative RFD (rRFD) during the initial phase of contraction (100 ms time interval relative to onset of contraction). Prior to disuse, marked age-related differences (p<0.05) were observed in isometric and dynamic muscle strength (~35%) as well as in RFD and Impulse (~39%). Following disuse, young and old individuals experienced comparable decrements (p<0.05) in isometric strength (~9%), slow dynamic strength (~13%), and RFD and Impulse (~19%), whereas old individuals only experienced decrements (p<0.05) in moderate dynamic strength (12%) and rRFD (~17%). Following recovery, all measures of mechanical muscle function were restored in young individuals compared to pre-disuse values, while isometric, slow and moderate dynamic muscle strength remained suppressed (p<0.05) in old individuals (~8%) along with a tendency to suppressed RFD100 (p=0.068). In conclusion, 4 days of lower limb disuse led to marked decrements in knee extensor mechanical muscle function in both young and old individuals, yet with greater decrements observed in moderate dynamic strength and rapid muscle force capacity in old individuals. While 7 days of recovery - including free ambulation, one test session and a single session of strength training - was sufficient to restore mechanical muscle function in young individuals, old individuals appeared to have an impaired ability to fully recover as evidenced by suppressed values of isometric and dynamic muscle strength and rapid muscle force capacity.

Distinct GAGE and MAGE-A expression during early human development indicate specific roles in lineage differentiation

BACKGROUND Expression of cancer/testis-associated proteins (CTAs) has traditionally been considered to be restricted to germ cells in normal tissues and to different types of malignancies. We have evaluated the potential role of CTAs in early human development. METHODS Using immunohistochemistry and RT-PCR, we investigated the expression of CTAs in differentiated human embryonic stem cells (hESC) and in late embryos and early fetuses. RESULTS We found that melanoma antigen A (MAGE-A) family members were expressed during differentiation of hESC to embryoid bodies and in teratomas, and overlapped with expression of the neuroectodermal markers beta-tubulin 3, Pax6 and nestin. A widespread expression of MAGE-A was also observed in neurons of the early developing central nervous system and peripheral nerves. G antigen (GAGE) expression was present in the early ectoderm of embryos, including cells of the ectodermal ring and apical epidermal ridge. Neuroectodermal cells in the floor plate and adjacent processes and endfeet of radial glial cells also expressed GAGE. In addition, GAGE family members were expressed in the peripheral adrenal cortex of 6-9-week-old embryos and fetuses, which specifically correlated with massive cellular proliferation and establishment of the definitive and fetal zones. Overlapping expression of MAGE-A and GAGE proteins occurred in migrating primordial germ cells. CONCLUSIONS Our results show that CTAs, in addition to their role in germ cells, may be involved in early development of various types of somatic cells, and suggest that they are implicated in specific differentiation processes.

Four days of muscle disuse impairs single fiber contractile function in young and old healthy men.

The purpose of the study was to investigate the effects of 4 days of disuse (knee brace) on contractile function of isolated vastus lateralis fibers (n=486) from 11 young (24.3±0.9 yrs) and 11 old (67.2±1.0 yrs) healthy men having comparable levels of physical activity. Prior to disuse single fiber specific force (maximal Ca(2+)-activated force per cross-sectional area) was lower in MHC I vs. IIa fibers (p<0.05) both in young (44%) and old (32%), and specific force of MHC IIa fibers was lower in old vs. young (19%, p<0.05). Further, Ca(2+) sensitivity was higher in MHC I vs. IIa fibers (p<0.05) in both age groups. Following disuse single fiber specific force decreased (p<0.05) in MHC I fibers in young (-19%) and old (-17%), and in MHC IIa fibers in young (-21%) but not in old (-11%; ns). The extent of these decreases did not differ between young and old. Ca(2+) sensitivity decreased (p<0.05) in MHC I fibers in old (-0.11 pCa units) but not in young (-0.08 pCa units; ns). The extent of these decreases was greater in old vs. young (p<0.05). In conclusion, 4 days of lower limb disuse led to marked impairments in single muscle fiber specific force independently of age, while changes in Ca(2+) sensitivity were dependent on age and MHC isoform composition. The present findings stress the importance of determining and implementing effective preventive and rehabilitative approaches for old individuals exposed to as little as 4 days of disuse.

Distribution of myogenic progenitor cells and myonuclei is altered in women with vs. those without chronically painful trapezius muscle.

It is hypothesized that repeated recruitment of low-threshold motor units is an underlying cause of chronic pain in trapezius myalgia. This study investigated the distribution of satellite cells (SCs), myonuclei, and macrophages in muscle biopsies from the trapezius muscle of 42 women performing repetitive manual work, diagnosed with trapezius myalgia (MYA; 44 ± 8 yr; mean ± SD) and 20 matched healthy controls (CON; 45 ± 9 yr). Our hypothesis was that muscle of MYA, in particular type I fibers, would demonstrate higher numbers of SCs, myonuclei, and macrophages compared with CON. SCs were identified on muscle cross sections by combined immunohistochemical staining for Pax7, type I myosin, and laminin, allowing the number of SCs associated with type I and II fibers to be determined. We observed a pattern of SC distribution in MYA previously only reported for individuals above 70 yr of age. Compared with CON, MYA demonstrated 19% more SCs per fiber associated with type I fibers (MYA 0.098 ± 0.039 vs. CON 0.079 ± 0.031; P < 0.05) and 40% fewer SCs associated with type II fibers (MYA 0.047 ± 0.017 vs. CON 0.066 ± 0.035; P < 0.05). The finding of similar numbers of macrophages between the two groups was not in line with our hypothesis and suggests that the elevated SC content of MYA was not due to heightened inflammatory cell contents, but rather to provide new myonuclei. The findings of greater numbers of SCs in type I fibers of muscle subjected to repeated low-intensity work support our hypothesis and provide new insight into stimuli capable of regulating SC content.

Muscle Glycogen Content Modifies SR Ca2 + Release Rate in Elite Endurance Athletes.

PURPOSE The aim of the present study was to investigate the influence of muscle glycogen content on sarcoplasmic reticulum (SR) function and peak power output (Wpeak) in elite endurance athletes. METHODS Fourteen highly trained male triathletes (VO2max = 66.5 ± 1.3 mL O2·kg·min), performed 4 h of glycogen-depleting cycling exercise (HRmean = 73% ± 1% of maximum). During the first 4 h of recovery, athletes received either water (H2O) or carbohydrate (CHO), separating alterations in muscle glycogen content from acute changes affecting SR function and performance. Thereafter, all subjects received CHO-enriched food for the remaining 20-h recovery period. RESULTS Immediately after exercise, muscle glycogen content and SR Ca release rate was reduced to 32% ± 4% (225 ± 28 mmol·kg dw) and 86% ± 2% of initial levels, respectively (P < 0.01). Glycogen markedly recovered after 4 h of recovery with CHO (61% ± 2% of preexercise) and SR Ca release rate returned to preexercise level. However, in the absence of CHO during the first 4 h of recovery, glycogen and SR Ca release rate remained depressed, with the normalization of both parameters at the end of the 24 h of recovery after receiving a CHO-enriched diet. Linear regression demonstrated a significant correlation between SR Ca release rate and muscle glycogen content (P < 0.01, r = 0.30). The 4 h of cycling exercise reduced Wpeak by 5.5%-8.9% at different cadences (P < 0.05), and Wpeak was normalized after 4 h of recovery with CHO, whereas Wpeak remained depressed (P < 0.05) after water provision. Wpeak was fully recovered after 24 h in both the H2O and the CHO group. CONCLUSION In conclusion, the present results suggest that low muscle glycogen depresses muscle SR Ca release rate, which may contribute to fatigue and delayed recovery of Wpeak 4 h postexercise.

Strength training increases the size of the satellite cell pool in type I and II fibres of chronically painful trapezius muscle in females

Non‐Technical Summary  Exercise has been shown to greatly reduce pain in female computer users suffering from neck pain (myalgia), but understanding of differences between healthy and myalgic muscle at the cellular level is poor. We report a large increase in muscle stem cell number in types I and II fibres with heavy strength training in this population. These findings contribute to understanding of the potential of exercise to influence the activity of cells found in the painful skeletal muscle of individuals suffering from myalgia.