MESP1 knock-down in human iPSC attenuates early vascular progenitor cell differentiation after completed primitive streak specification.

MESP1 is a key transcription factor in development of early cardiovascular tissue and it is required for induction of the cardiomyocyte (CM) gene expression program, but its role in vascular development is unclear. Here, we used inducible CRISPRi knock-down of MESP1 to analyze the molecular processes of the early differentiation stages of human induced pluripotent stem cells into mesoderm and subsequently vascular progenitor cells. We found that expression of the mesodermal marker, BRACHYURY (encoded by T) was unaffected in MESP1 knock-down cells as compared to wild type cells suggesting timely movement through the primitive streak whereas another mesodermal marker MIXL1 was slightly, but significantly decreased. In contrast, the expression of the vascular cell surface marker KDR was decreased and CD31 and CD34 expression were substantially reduced in MESP1 knock-down cells supporting inhibition or delay of vascular specification. In addition, mRNA microarray data revealed several other altered gene expressions including the EMT regulating transcription factors SNAI1 and TWIST1, which were both significantly decreased indicating that MESP1 knock-down cells are less likely to undergo EMT during vascular progenitor differentiation. Our study demonstrates that while leaving primitive streak markers unaffected, MESP1 expression is required for timely vascular progenitor specification. Thus, MESP1 expression is essential for the molecular features of early CM, EC and VSMC lineage specification.

Stem cell survival is severely compromised by the thymidineanalog EdU (5-ethynyl-2'-deoxyuridine), an alternative to BrdU for proliferation assays and stem cell tracing.

Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2'-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation into regenerating muscle, whereas stem cells labeled in parallel with another dye survived very well and also participated in myofiber formation. Similar data were obtained upon in vitro myogenic culture, and further analysis showed that EdU reduced cell numbers by up to 88 % and increased the cell volume of remaining cells by as much as 91 %. Even at low EdU concentrations, cell survival and phenotype were substantially compromised, and the myogenic differentiation potential was inhibited. Since we examined both primary derived cells and cell lines from several species with the same result, this appears to be a common trait of EdU. We therefore suggest that EdU labeling should be avoided (or used with precaution) for stem cell tracing purposes.

Angiotensin II regulates microRNA-132/-212 in hypertensive rats and humans.

MicroRNAs (miRNAs), a group of small non-coding RNAs that fine tune translation of multiple target mRNAs, are emerging as key regulators in cardiovascular development and disease. MiRNAs are involved in cardiac hypertrophy, heart failure and remodeling following cardiac infarction; however, miRNAs involved in hypertension have not been thoroughly investigated. We have recently reported that specific miRNAs play an integral role in Angiotensin II receptor (AT1R) signaling, especially after activation of the Gαq signaling pathway. Since AT1R blockers are widely used to treat hypertension, we undertook a detailed analysis of potential miRNAs involved in Angiotensin II (AngII) mediated hypertension in rats and hypertensive patients, using miRNA microarray and qPCR analysis. The miR-132 and miR-212 are highly increased in the heart, aortic wall and kidney of rats with hypertension (159 ± 12 mm Hg) and cardiac hypertrophy following chronic AngII infusion. In addition, activation of the endothelin receptor, another Gαq coupled receptor, also increased miR-132 and miR-212. We sought to extend these observations using human samples by reasoning that AT1R blockers may decrease miR-132 and miR-212. We analyzed tissue samples of mammary artery obtained from surplus arterial tissue after coronary bypass operations. Indeed, we found a decrease in expression levels of miR-132 and miR-212 in human arteries from bypass-operated patients treated with AT1R blockers, whereas treatment with ß-blockers had no effect. Taken together, these data suggest that miR-132 and miR-212 are involved in AngII induced hypertension, providing a new perspective in hypertensive disease mechanisms.

Delta-like 1/fetal antigen-1 (Dlk1/FA1) is a novel regulator of chondrogenic cell differentiation via inhibition of the Akt kinase-dependent pathway.

Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis.

MicroRNA-15a fine-tunes the level of Delta-like 1 homolog (DLK1) in proliferating 3T3-L1 preadipocytes.

Delta like 1 homolog (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal; however, little is known about the underlying mechanisms. We here report that miR-15a modulates DLK1 levels in preadipocytes thus providing a mechanism for DLK1 regulation that further links it to cell cycle arrest and cancer since miR-15a is deregulated in these processes. In preadipocytes, miR-15a increases with cell density, and peaks at the same stage where membrane DLK1(M) and soluble DLK1(S) are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1(M) protein while it increases the amount of DLK1(S) supporting a direct repression of DLK1 and a parallel effect on the protease that cleaves off the DLK1 from the membrane. In agreement with previous studies, we found that miR-15a represses cell numbers, but additionally, we report that miR-15a also increases cell size. Conversely, anti-miR-15a treatment decreases cell size while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1(S). Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling networks.

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy.

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC's as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC's is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Characterization of DLK1+ cells emerging during skeletal muscle remodeling in response to myositis, myopathies, and acute injury.

Delta like 1 (DLK1) has been proposed to act as a regulator of cell fate determination and is linked to the development of various tissues including skeletal muscle. Herein we further investigated DLK1 expression during skeletal muscle remodeling. Although practically absent in normal adult muscle, DLK1 was upregulated in all human myopathies analyzed, including Duchenne- and Becker muscular dystrophies. Substantial numbers of DLK1(+) satellite cells were observed in normal neonatal and Duchenne muscle, and furthermore, myogenic DLK1(+) cells were identified during muscle regeneration in animal models in which the peak expression of Dlk1 mRNA and protein coincided with that of myoblast differentiation and fusion. In addition to perivascular DLK1(+) cells, interstitial DLK1(+) cells were numerous in regenerating muscle, and in agreement with colocalization studies of DLK1 and CD90/DDR2, qPCR of fluorescence-activated cell sorting DLK1(+) and DLK1(-) cells revealed that the majority of DLK1(+) cells isolated at day 7 of regeneration had a fibroblast-like phenotype. The existence of different DLK1(+) populations was confirmed in cultures of primary derived myogenic cells, in which large flat nonmyogenic DLK1(+) cells and small spindle-shaped cells coexpressing DLK1 and muscle-specific markers were observed. Myogenic differentiation was achieved when sorted DLK1(+) cells were cocultured together with primary myoblasts revealing a myogenic potential that was 10% of the DLK1(-) population. Transplantation of DLK1(+) cells into lacerated muscle did, however, not give rise to DLK1(+) cell-derived myofibers. We suggest that the DLK1(+) subpopulations identified herein each may contribute at different levels/time points to the processes involved in muscle development and remodeling.

Secreted protein acidic and rich in cysteine (SPARC) in human skeletal muscle.

Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15-16 weeks. SPARC protein was detected in the majority of analyzed muscle biopsies (23 of 24), mainly in mononuclear cells of which few were pax7 positive. Myotubes and regenerating myofibers also expressed SPARC. The expression-degree seemed to reflect the severity of the lesion. In accordance with these in vivo findings, primary human-derived satellite cells were found to express SPARC both during proliferation and differentiation in vitro. In conclusion, this study shows SPARC expression both during muscle development and in regenerating muscle. The expression is detected both in satellite cells/myoblasts and in myotubes and muscle fibers, indicating a role for SPARC in the skeletal muscle compartment.

Non-cultured adipose-derived CD45- side population cells are enriched for progenitors that give rise to myofibres in vivo.

Side population (SP) cells are highly able to exclude the Hoechst 33342 dye through membrane transporters, a feature associated with cell immaturity and therefore proposed as a marker of stem cells. Herein we demonstrate that the adipose tissue derived stromal vascular fraction (SVF) contains a novel population of non-haematopoietic "side population" (SPCD45(-)) cells. Simultaneous qRT-PCR of 64 genes revealed that the freshly isolated SPCD45(-) was highly enriched for cells expressing genes related to stem cells, the Notch pathway, and early vascular precursors. Notably, the expression of smooth muscle actin, C-met and Cd34 together with Angpt2, Flk1, VE-cadherin, and Cd31 suggested a phenotypic resemblance to pericytes and aorta-derived mesoangioblasts. Recent evidence suggests that cells residing within the vascular niche may participate in regeneration of skeletal muscle and although skeletal muscle repair mainly relies on the satellite cell, several reports have shown that vessel-associated cells may adopt a myogenic phenotype when exposed to a muscle environment. In accordance with these findings, we also observed in vitro myogenic specification of SPCD45(-) cells when cocultured with myoblasts. Furthermore, immediate intramuscular engraftment of non-cultured SPCD45(-) cells gave rise to myofibres and cells lining blood vessels, whereas the SVF only provided donor derived mononuclear cells. We therefore conclude that the SPCD45(-) fraction of adipose-derived SVF is enriched for cells expressing vascular associated markers and that the myogenic differentiation potential of these cells does not depend on prior in vitro expansion.

Antibody-based inhibition of circulating DLK1 protects from estrogen deficiency-induced bone loss in mice.

Soluble delta-like 1 homolog (DLK1) is a circulating protein that belongs to the Notch/Serrate/delta family, which regulates many differentiation processes including osteogenesis and adipogenesis. We have previously demonstrated an inhibitory effect of DLK1 on bone mass via stimulation of bone resorption and inhibition of bone formation. Further, serum DLK1 levels are elevated and positively correlated to bone turnover markers in estrogen (E)-deficient rodents and women. In this report, we examined whether inhibition of serum DLK1 activity using a neutralizing monoclonal antibody protects from E deficiency-associated bone loss in mice. Thus, we generated mouse monoclonal anti-mouse DLK1 antibodies (MAb DLK1) that enabled us to reduce and also quantitate the levels of bioavailable serum DLK1 in vivo. Ovariectomized (ovx) mice were injected intraperitoneally twice weekly with MAb DLK1 over a period of one month. DEXA-, microCT scanning, and bone histomorphometric analyses were performed. Compared to controls, MAb DLK1 treated ovx mice were protected against ovx-induced bone loss, as revealed by significantly increased total bone mass (BMD) due to increased trabecular bone volume fraction (BV/TV) and inhibition of bone resorption. No significant changes were observed in total fat mass or in the number of bone marrow adipocytes. These results support the potential use of anti-DLK1 antibody therapy as a novel intervention to protect from E deficiency associated bone loss.

Dlk1/FA1 is a novel endocrine regulator of bone and fat mass and its serum level is modulated by growth hormone.

Fat and bone metabolism are two linked processes regulated by several hormonal factors. Fetal antigen 1 (FA1) is the soluble form of dlk1 (delta-like 1), which is a member of the Notch-Delta family. We previously identified FA1 as a negative regulator of bone marrow mesenchymal stem cell differentiation. Here, we studied the effects of circulating FA1 on fat and bone mass in vivo by generating mice expressing high serum levels of FA1 (FA1 mice) using the hydrodynamic-based gene transfer procedure. We found that increased serum FA1 levels led to a significant reduction in total body weight, fat mass, and bone mass in a dose-dependent manner. Reduced bone mass in FA1 mice was associated with the inhibition of mineral apposition rate and bone formation rates by 58 and 72%, respectively. Because FA1 is colocalized with GH in the pituitary gland, we explored the possible modulation of serum FA1 by GH. Serum levels of IGF-I and IGF binding proteins did not change in FA1 mice, whereas increasing serum GH in normal mice using hydrodynamic-based gene transfer procedure dramatically reduced serum FA1 levels by 60%. Conversely, serum FA1 was increased 450% in hypophysectomized mice, and this high level was reduced by 40% during GH treatment. In conclusion, our data identify the FA1 as a novel endocrine factor regulating bone mass and fat mass in vivo, and its serum levels are regulated by GH. FA1 thus provides a novel class of developmental molecules that regulate physiological functions of the postnatal organisms.

The influence of anti-inflammatory medication on exercise-induced myogenic precursor cell responses in humans.

The consumption of nonsteroidal anti-inflammatory drugs (NSAIDs) is widespread among athletes when faced with muscle soreness or injury, but the effects of NSAIDs on satellite cell activity in humans are unknown. To investigate this, 14 healthy male endurance athletes (mean peak oxygen consumption 62 ml x kg(-1) x min(-1)) volunteered for the study, which involved running 36 km. They were divided into two groups and received either 100 mg indomethacin per day or placebo. Muscle biopsies collected before the run and on days 1, 3, and 8 afterward were analyzed for satellite cells by immunohistochemistry with the aid of neural cell adhesion molecule (NCAM) and fetal antigen-1 (FA1) antibodies. Muscle biopsies were also collected from untrained individuals for comparison. Compared with preexercise levels, a 27% increase in the number of NCAM+ cells was observed on day 8 postexercise in the placebo group (P < 0.05), while levels remained similar at all time points in the NSAID group. No change was seen in the proportion of FA1+ cells, although lower levels were found in the muscle of endurance-trained athletes compared with untrained individuals (P < 0.05). These results suggest that ingestion of anti-inflammatory drugs attenuates the exercise-induced increase in satellite cell number, supporting the role of the cyclooxygenase pathway in satellite cell activity.

Cardiac injury of the newborn mammalian heart accelerates cardiomyocyte terminal differentiation.

After birth cardiomyocytes undergo terminal differentiation, characterized by binucleation and centrosome disassembly, rendering the heart unable to regenerate. Yet, it has been suggested that newborn mammals regenerate their hearts after apical resection by cardiomyocyte proliferation. Thus, we tested the hypothesis that apical resection either inhibits, delays, or reverses cardiomyocyte centrosome disassembly and binucleation. Our data show that apical resection rather transiently accelerates centrosome disassembly as well as the rate of binucleation. Consistent with the nearly 2-fold increased rate of binucleation there was a nearly 2-fold increase in the number of cardiomyocytes in mitosis indicating that the majority of injury-induced cardiomyocyte cell cycle activity results in binucleation, not proliferation. Concurrently, cardiomyocytes undergoing cytokinesis from embryonic hearts exhibited midbody formation consistent with successful abscission, whereas those from 3 day-old cardiomyocytes after apical resection exhibited midbody formation consistent with abscission failure. Lastly, injured hearts failed to fully regenerate as evidenced by persistent scarring and reduced wall motion. Collectively, these data suggest that should a regenerative program exist in the newborn mammalian heart, it is quickly curtailed by developmental mechanisms that render cardiomyocytes post-mitotic.

Evidence of non-canonical NOTCH signaling: Delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals.

Canonical NOTCH signaling, known to be essential for tissue development, requires the Delta-Serrate-LAG2 (DSL) domain for NOTCH to interact with its ligand. However, despite lacking DSL, Delta-like 1 homolog (DLK1), a protein that plays a significant role in mammalian development, has been suggested to interact with NOTCH1 and act as an antagonist. This non-canonical interaction is, however controversial, and evidence for a direct interaction, still lacking in mammals. In this study, we elucidated the putative DLK1-NOTCH1 interaction in a mammalian context. Taking a global approach and using Dlk1(+/+) and Dlk1(-/-) mouse tissues at E16.5, we demonstrated that several NOTCH signaling pathways indeed are affected by DLK1 during tissue development, and this was supported by a lower activation of NOTCH1 protein in Dlk1(+/+) embryos. Likewise, but using a distinct Dlk1-manipulated (siRNA) setup in a mammalian cell line, NOTCH signaling was substantially inhibited by DLK1. Using a mammalian two-hybrid system, we firmly established that the effect of DLK1 on NOTCH signaling was due to a direct interaction between DLK1 and NOTCH1. By careful dissection of this mechanism, we found this interaction to occur between EGF domains 5 and 6 of DLK1 and EGF domains 10-15 of NOTCH1. Thus, our data provide the first evidence for a direct interaction between DLK1 and NOTCH1 in mammals, and substantiate that non-canonical NOTCH ligands exist, adding to the complexity of NOTCH signaling.

Neonatal epicardial-derived progenitors aquire myogenic traits in skeletal muscle, but not cardiac muscle.

BACKGROUND/OBJECTIVES: Epicardium-derived progenitor cells (EPDCs) differentiate into all heart cell types in the embryonic heart, yet their differentiation into cardiomyocytes in the adult heart is limited and poorly described. This may be due to EPDCs lacking myogenic potential or the inert adult heart missing regenerative signals essential for directed differentiation of EPDCs. Herein, we aimed to evaluate the myogenic potential of neonatal EPDCs in adult and neonatal mouse myocardium, as well as in skeletal muscle. The two latter tissues have an intrinsic capability to develop and regenerate, in contrast to the adult heart. METHODS: Highly purified mouse EPDCs were transplanted into damaged neonatal and adult myocardium as well as regenerating skeletal muscle. Co-cultures with skeletal myoblasts were used to distinguish fusion independent myogenic conversion. RESULTS: No donor EPDC-derived cardiomyocytes were observed in hearts. In contrast, a remarkable contribution of EPDCs to skeletal muscle myofiber formation was evident in vivo. Furthermore, co-cultures of EPDCs with myoblasts showed that EPDCs became part of multinucleated fibers and appeared to acquire myogenic traits independent of a fusion event. Fluorescence activated cell sorting of EPDCs co-cultured with and without myoblasts and subsequent qRT-PCR of 64 transcripts established that the myogenic phenotype conversion was accomplished through induction of a transcriptional myogenic program. CONCLUSION: These results suggest that EPDCs may be more myogenic than previously anticipated. But, the heart may lack factors for induction of myogenesis of EPDCs, a scenario that should be taken into consideration when aiming for repair of damaged myocardium by stem cell transplantation.

Characterization of fetal antigen 1/delta-like 1 homologue expressing cells in the rat nigrostriatal system: effects of a unilateral 6-hydroxydopamine lesion.

Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc dopaminergic neurons and/or due to the stab wound. Our findings hint to a significant role of FA1/dlk1 in the SNc during early postnatal development. The differential expression of FA1/dlk1 in the SNc and the striatum of dopamine-depleted rats could indicate a potential involvement of FA1/dlk1 in the cellular response to the degenerative processes.

Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1.

UNLABELLED: Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARgamma2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor -1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected. CONCLUSION: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.

Enhanced procollagen processing in skeletal muscle after a single bout of eccentric loading in humans.

Increases in procollagen processing within skeletal muscle have previously been reported in small animal models only. While indirect measurements in humans have suggested an increase procollagen processing, no intra-skeletal muscle measurements have confirmed these findings. In this study, eight young healthy male subjects performed a single bout of unaccustomed high intensity eccentric exercise on one leg, with the contralateral leg being the control. A significant increase in the muscle interstitial concentration of the N-terminal propeptide of procollagen type I (PINP) was observed (day 0: 1.96 +/- 0.44 ng ml(-1), day 2: 1.94 +/- 0.32 ng ml(-1), day 4: 3.90 +/- 1.03 ng ml(-1), day 8: 7.23 +/- 2.34 ng ml(-1)*, *P < 0.05 vs. basal and control) with no change being noted in the control leg. By day 2 post-exercise, an increase in the histological immunoreactivity of PINP and the N-terminal propeptide of procollagen type III (PIIINP) was also shown in the exercising leg only. Further, from day 2 post-exercise, immunoreactivity for tenascin C and reactive macrophages (CD68+ cells) was seen within the perimysial and endomysial connective tissue of the exercising leg only, indicating a high mechanical load and inflammation. This study shows that following a single bout of high intensity eccentric exercise there is an increase in procollagen processing within skeletal muscle in humans.

Horse serum reduces expression of membrane-bound and soluble isoforms of the preadipocyte marker Delta-like 1 homolog (Dlk1), but is inefficient for adipogenic differentiation of mouse preadipocytes.

Downregulation of the preadipocyte marker Delta-like 1 homologue (Dlk1), an inhibitor of adipogenesis, has been suggested to be a prerequisite for adipogenic differentiation to occur, and low Dlk1 levels are often used to verify adipogenesis. Mouse preadipocytic cell lines such as 3T3-L1, as well as primary derived preadipocytes, are important models to study adipogenic differentiation and obesity. However, in vitro adipogenic differentiation of primary derived preadipocytes remains incomplete, and identification of factors that will improve the adipogenic differentiation process is thus of high value. In this study we show that horse serum fails to improve adipogenic differentiation of mouse preadipocytes (both 3T3-L1 cells and primary derived mouse preadipocytes) as otherwise reported for bone marrow derived adipogenic precursors. Unexpectedly, while Dlk1 levels were indeed decreased using horse serum, this did not correlate with a high degree of adipogenic differentiation. In conclusion, our novel results thus reveal that horse serum clearly is insufficient for adipogenic differentiation of mouse preadipocytes and that low levels of Dlk1 alone are a poor marker of mouse in vitro adipogenesis. We would also like to emphasize that it is very important for the field of cellular differentiation that researchers thoroughly investigate the effect of individual reagents in their protocols. Such data will increase understanding of the limitations and possibilities of individual systems.

Preadipocytes proliferate and differentiate under the guidance of Delta-like 1 homolog (DLK1).

Obesity occurs when an excessive dietary fat intake leads to expansion of adipose tissue, which mainly consists of adipocytes that arise from proliferating and differentiating adipose stem cells, the preadipocytes. Obesity is a consequence of both adipocyte hypertrophy and hyperplasia. Knowledge about preadipocyte differentiation is relatively well established, whereas the mechanism responsible for preadipocyte proliferation is incompletely understood and only in the early stage of comprehension. In this regard, we have recently identified that Delta-like 1 homolog (Dlk1) (also known as Preadipocyte factor 1 [Pref-1]) inhibits preadipocyte proliferation by regulating their entry into G1/S-phase. This novel disclosure, adding to the previous published data on Dlk1 repression of preadipocyte differentiation, has given us the chance to firmly place Dlk1 as a master regulator of preadipocyte homeostasis and adipose tissue expansion. Dlk1 manipulation may, therefore, open new perspectives in obesity treatments.