Muscle origin of porcine satellite cells affects in vitro differentiation potential

2010 ◽  
Vol 28 (5) ◽  
pp. 403-411 ◽  
Author(s):  
Zoe Redshaw ◽  
Steven McOrist ◽  
Paul Loughna
Keyword(s):  
Satellite Cells ◽  
In Vitro Differentiation ◽  
Differentiation Potential ◽  
Muscle Origin

Rat and mouse epiblasts differ in their capacity to generate extraembryonic endoderm

1998 ◽  
Vol 10 (8) ◽  
pp. 517 ◽  
Author(s):  
Jennifer Nichols ◽  
Austin Smith ◽  
Mia Buehr
Keyword(s):  
In Vitro Differentiation ◽  
Differentiation Potential ◽  
Rat Embryos ◽  
Extraembryonic Endoderm ◽  
Parietal Endoderm

In this study we have compared the in vitro differentiation potential of epiblast tissue from mouse and rat embryos. Epiblasts were isolated from egg cylinder stage embryos by microdissection and placed in culture. Rat cultures were distinguished by the copious production of parietal endoderm cells. Mouse epiblasts, in contrast, did not produce parietal endoderm. This difference in capacity to regenerate extraembryonic endoderm marks a surprising distinction in development of the pluripotential lineage between these two closely related rodents.

296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Mohana Kumar ◽  
W. J. Lee ◽  
Y. M. Lee ◽  
R. Patil ◽  
S. L. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Class Ii ◽  
In Vitro Differentiation ◽  
Rt Pcr ◽  
Hepatic Differentiation ◽  
Alizarin Red ◽  
Differentiation Potential

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

scholarly journals Generation of in vivo neural stem cells using partially reprogrammed cells defective in in vitro differentiation potential

Oncotarget ◽  
2017 ◽  
Vol 8 (10) ◽  
pp. 16456-16462 ◽  
Author(s):  
Jong Soo Kim ◽  
Yean Ju Hong ◽  
Hyun Woo Choi ◽  
Hyuk Song ◽  
Sung June Byun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
In Vitro Differentiation ◽  
Differentiation Potential

scholarly journals Modulation of HOXA9 after skeletal muscle denervation and reinnervation

2020 ◽  
Author(s):  
Xiaomei Lu ◽  
Bingsheng Liang ◽  
Shuaijie Li ◽  
Zhi Chen ◽  
Wenkai Chang
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Satellite Cells ◽  
Gastrocnemius Muscle ◽  
Myogenic Differentiation ◽  
Cell Activity ◽  
Sham Operation ◽  
Differentiation Potential ◽  
Stem Cell Activity

Abstract Background HOXA9 (Homeobox A9), whose expression is promoted by MLL1 (Mixed Lineage Leukemia 1) and WDR5 (WD-40 repeat protein 5), is a homeodomain-containing transcription factor which plays an essential role in regulating stem cell activity. HOXA9 inhibits regeneration of skeletal muscle and delays the recovery after muscle wound in aged mice, but is little known in denervated/reinnervated muscles. Methods we performed detailed time-process expression analysis on HOXA9 and its promotors, MLL1 and WDR5, in the rat gastrocnemius muscle after three types of sciatic nerve surgeries: nerve transection (denervation); end-to-end repairing (repairing); and the sham operation. Then the specific mechanisms of Hoxa9 were detected in vitro through primary satellite cells transfected respectively by pIRES2-DsRed2 empty plasmids, pIRES2-DsRed2-HOXA9 plasmids, pPLK/ GFP -Puro empty plasmids, and pPLK/GFP-Puro- HOXA9 shRNA plasmids. Results We found that HOXA9 expression was synchronous with the severity of muscle atrophy, as well as the upregulation of MLL1 and WDR5 associated with the denervation state to some extent. Indeed, experiments with primary satellite cells revealed that HOXA9 inhibited myogenic differentiation, but not destroy the differentiation potential, influenced the best-known atrophic pathways, and promoted apoptosis. Conclusion HOXA9 may play a pro-atrophic role in denervated muscle atrophy.

scholarly journals A Novel Approach for the Isolation and Long-Term Expansion of Pure Satellite Cells Based on Ice-Cold Treatment

2020 ◽  
Author(s):  
Anna Benedetti ◽  
Gianluca Cera ◽  
Daniele De Meo ◽  
Ciro Villani ◽  
Marina Bouche ◽  
...  
Keyword(s):  
Satellite Cells ◽  
Mononuclear Cells ◽  
Cold Treatment ◽  
Cell Manipulation ◽  
Minimal Cell ◽  
Differentiation Potential ◽  
Novel Approach ◽  
In Vitro Expansion

Abstract Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15-30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.

scholarly journals Modulation of HOXA9 after skeletal muscle denervation and reinnervation

2020 ◽  
Vol 318 (6) ◽  
pp. C1154-C1165
Author(s):  
Xiaomei Lu ◽  
Bingsheng Liang ◽  
Shuaijie Li ◽  
Zhi Chen ◽  
Wenkai Chang
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Satellite Cells ◽  
Myogenic Differentiation ◽  
Cell Activity ◽  
Skeletal Muscle Regeneration ◽  
Sham Operation ◽  
Denervated Muscle ◽  
Differentiation Potential

Homeobox A9 (HOXA9), the expression of which is promoted by mixed lineage leukemia 1 (MLL1) and WD-40 repeat protein 5 (WDR5), is a homeodomain-containing transcription factor that plays an essential role in regulating stem cell activity. HOXA9 has been found to inhibit skeletal muscle regeneration and delay recovery after muscle wounding in aged mice, but little is known about its role in denervated/reinnervated muscles. We performed detailed time-dependent expression analyses of HOXA9 and its promoters, MLL1 and WDR5, in rat gastrocnemius muscles after the following three types of sciatic nerve surgeries: nerve transection (denervation), end-to-end repair (repair), and sham operation (sham). Then, the specific mechanisms of HOXA9 were detected in vitro by transfecting primary satellite cells with empty pIRES2-DsRed2, pIRES2-DsRed2-HOXA9, empty pPLK/GFP-Puro, and pPLK/GFP-Puro-HOXA9 small hairpin RNA (shRNA) plasmids. We found, for the first time, that HOXA9 protein expression simultaneously increased with increasing denervated muscle atrophy severity and that upregulated MLL1 and WDR5 expression was partly associated with denervation. Indeed, in vitro experiments revealed that HOXA9 inhibited myogenic differentiation, affected the best known atrophic signaling pathways, and promoted apoptosis but did not eliminate the differentiation potential of primary satellite cells. HOXA9 may promote denervated muscle atrophy by regulating the activity of satellite cells.

scholarly journals A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Benedetti ◽  
Gianluca Cera ◽  
Daniele De Meo ◽  
Ciro Villani ◽  
Marina Bouche ◽  
...  
Keyword(s):  
Satellite Cells ◽  
Mononuclear Cells ◽  
Cold Treatment ◽  
Cell Manipulation ◽  
Minimal Cell ◽  
Differentiation Potential ◽  
Novel Approach ◽  
In Vitro Expansion

AbstractSatellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15–30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.

Dynamic distribution and formation of a para-sarcomeric banding pattern of prosomes during myogenic differentiation of satellite cells in vitro

1999 ◽  
Vol 112 (7) ◽  
pp. 989-1001 ◽  
Author(s):  
J. Foucrier ◽  
M.C. Grand ◽  
F. De Conto ◽  
Y. Bassaglia ◽  
G. Geraud ◽  
...  
Keyword(s):  
Satellite Cells ◽  
Banding Pattern ◽  
Myogenic Differentiation ◽  
In Vitro Differentiation ◽  
Adult Skeletal Muscle ◽  
Myogenic Stem Cells ◽  
Alpha Actin ◽  
Type Intermediate

Myogenesis proceeds by fusion of proliferating myoblasts into myotubes under the control of various transcription factors. In adult skeletal muscle, myogenic stem cells are represented by the satellite cells which can be cultured and differentiate in vitro. This system was used to investigate the subcellular distribution of a particular type of prosomes at different steps of the myogenic process. Prosomes constitute the MCP core of the 26S proteasomes but were first observed as subcomplexes of the untranslated mRNPs; recently, their RNase activity was discovered. A monoclonal antibody raised against the p27K subunit showed that the p27K subunit-specific prosomes move transiently into the nucleus prior to the onset of myoblast fusion into myotubes; this represents possibly one of the first signs of myoblast switching into the differentiation pathway. Prior to fusion, the prosomes containing the p27K subunit return to the cytoplasm, where they align with the gradually formed lengthwise-running desmin-type intermediate filaments and the microfilaments, co-localizing finally with the actin bundles. The prosomes progressively form discontinuous punctate structures which eventually develop a pseudo-sarcomeric banding pattern. In myotubes just formed in vitro, the formation of this pattern seems to preceed that produced by the muscle-specific sarcomeric (alpha)-actin. Interestingly, this pattern of prosomes of myotubes in terminal in vitro differentiation was very similar to that of prosomes observed in vivo in foetal and adult muscle. These observations are discussed in relation to molecular myogenesis and prosome/proteasome function.

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