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.

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.

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.

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.

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.

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.

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.

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.

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.

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 homolog (dlk1): a marker for rhabdomyosarcomas implicated in skeletal muscle regeneration.

Dlk1, a member of the Epidermal Growth Factor family, is expressed in multiple tissues during development, and has been detected in carcinomas and neuroendocrine tumors. Dlk1 is paternally expressed and belongs to a group of imprinted genes associated with rhabdomyosarcomas but not with other primitive childhood tumors to date. Here, we investigate the possible roles of Dlk1 in skeletal muscle tumor formation. We analyzed tumors of different mesenchymal origin for expression of Dlk1 and various myogenic markers and found that Dlk1 was present consistently in myogenic tumors. The coincident observation of Dlk1 with a highly proliferative state in myogenic tumors led us to subsequently investigate the involvement of Dlk1 in the control of the adult myogenic programme. We performed an injury study in Dlk1 transgenic mice, ectopically expressing ovine Dlk1 (membrane bound C2 variant) under control of the myosin light chain promotor, and detected an early, enhanced formation of myotubes in Dlk1 transgenic mice. We then stably transfected the mouse myoblast cell line, C2C12, with full-length Dlk1 (soluble A variant) and detected an inhibition of myotube formation, which could be reversed by adding Dlk1 antibody to the culture supernatant. These results suggest that Dlk1 is involved in controlling the myogenic programme and that the various splice forms may exert different effects. Interestingly, both in the Dlk1 transgenic mice and the DLK1-C2C12 cells, we detected reduced myostatin expression, suggesting that the effect of Dlk1 on the myogenic programme might involve the myostatin signaling pathway. In support of a relationship between Dlk1 and myostatin we detected reciprocal expression of these two transcripts during different cell cycle stages of human myoblasts. Together our results suggest that Dlk1 is a candidate marker for skeletal muscle tumors and might be involved directly in skeletal muscle tumor formation through a modulatory effect on the myogenic programme.

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.

Membrane-tethered delta-like 1 homolog (DLK1) restricts adipose tissue size by inhibiting preadipocyte proliferation.

Adipocyte renewal from preadipocytes has been shown to occur throughout life and to contribute to obesity, yet very little is known about the molecular circuits that control preadipocyte expansion. The soluble form of the preadipocyte factor (also known as pref-1) delta-like 1 homolog (DLK1(S)) is known to inhibit adipogenic differentiation; however, the impact of DLK1 isoforms on preadipocyte proliferation remains to be determined. We generated preadipocytes with different levels of DLK1 and examined differentially affected gene pathways, which were functionally tested in vitro and confirmed in vivo. Here, we demonstrate for the first time that only membrane-bound DLK1 (DLK1(M)) exhibits a substantial repression effect on preadipocyte proliferation. Thus, by independently manipulating DLK1 isoform levels, we established that DLK1(M) inhibits G1-to-S-phase cell cycle progression and thereby strongly inhibits preadipocyte proliferation in vitro. Adult DLK1-null mice exhibit higher fat amounts than wild-type controls, and our in vivo analysis demonstrates that this may be explained by a marked increase in preadipocyte replication. Together, these data imply a major dual inhibitory function of DLK1 on adipogenesis, which places DLK1 as a master regulator of preadipocyte homeostasis, suggesting that DLK1 manipulation may open new avenues in obesity treatment.

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.

Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells.

Human mesenchymal stem cells (hMSC) are currently being introduced for cell therapy, yet, antibodies specific for native and differentiated MSCs are required for their identification prior to clinical use. Herein, high quality antibodies against MSC surface proteins were developed by immunizing mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic differentiation. Interestingly, undifferentiated cells revealed a sole cytoplasmic distribution pattern of Collagen VI, which however changed to an extracellular matrix appearance upon osteogenic- and adipogenic differentiation. In relation to this, we found that STRO-1(+/-)/Collagen VI(-) sorted hMSC contained fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs, and in addition our results may suggest that the DJ18 generated antibody against Collagen VI can be used for negative selection of cultured undifferentiated MSCs.

Newly formed skeletal muscle fibers are prone to false positive immunostaining by rabbit antibodies.

Reports on muscle biology and regeneration often implicate immuno(cyto/histo)chemical protein characterization using rabbit polyclonal antibodies. In this study we demonstrate that newly formed myofibers are especially prone to false positive staining by rabbit antibodies and this unwanted staining is only recognized (1) by a negative muscle tissue control that does not harbor the protein to be examined (fx. from knockout mouse) or (2) by use of a nonsense rabbit antibody that has been prepared in the same way as the antibody of interest. However, many muscle immuno(cyto/histo)chemical studies only rely on controls that reveal non-specific binding by the secondary antibody and neglect that the primary rabbit antibody itself may cause false positive staining of the muscle. We suggest that reliable immuno-based protein detection in newly formed muscle fibers at least requires a nonsense rabbit antibody and optimally a negative muscle/cell control.

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.

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.