Isolation of Skeletal Muscle-derived Cells Modeling Neural Crest-Derived Stem Cells for Therapeutic Use in Regenerative Periodontology

Periodontitis is microbial infection affecting periodontium, the tooth supporting structure and affects >743 million people worldwide. Neural crest-derived stem cells (NCSCs) hold the promise to regenerate the damaged periodontium. These cells have been identified within adult adipose tissue, periodontal ligament, and palatal tissue. Typical enzymatic isolation protocols are expensive, time consuming and often not clinically compliant. Enzyme-free, mechanical dissociation has been suggested as an alternative method of generating cell suspensions required for cell separation and subsequent expansion ex vivo. In our study, samples of rat skeletal muscle tissue were used to appraise the suitability of a novel mincing method of mechanical dissociation against enzymatic digestion with collagenase and dispase. Skeletal muscle is readily available and has been shown to contain NCSC populations. We used a Rigenera-Human Brain Wave® prototype mincer to produce a suspension of skeletal muscle-derived cells modeling NCSCs. We have compared the resulting cell cultures produced via mechanical dissociation and enzymatic dissociation, producing single cell suspensions suitable for Magnetic Cell Sorting (MACs) and Fluorescence-activated cell sorting (FACS). Despite the Countess Automated Cytometry data demonstrating that cell suspensions produced by mechanical dissociation (n=24) contain on average 26.8 times as many viable cells as enzymatic cell suspensions (n=18), enzymatic suspensions produced more successful cell cultures. Spheroids and subsequently adherent cells formed from 4 enzymatic cell suspensions (44.4%) vs. 1 mechanical cell suspension (8.3%). Enzymatic digestion protocols formed spheroids faster and more plentifully than mechanical cell suspensions. Adherent cells and spheroids isolated via both methods appear morphologically similarly to NCSCs from our previous studies.

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Isolation of Neural Crest-derived Stem Cells for Therapeutic Use in Regenerative Periodontology

10.20944/preprints202005.0427.v1 ◽

2020 ◽

Author(s):

Wolf-Dieter Grimm ◽

Nikolaj Didenko5 ◽

Kunaal Dhingra ◽

Alexander Dolgalev ◽

Natella Enukashvily ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Neural Crest ◽

Cell Cultures ◽

Cell Sorting ◽

Enzymatic Digestion ◽

Cell Suspensions ◽

Adherent Cells ◽

Brain Wave ◽

Mechanical Dissociation

Periodontitis is microbial infection affecting periodontium, the tooth supporting structure and affects >743 million people worldwide. Neural crest-derived stem cells (NCSCs) hold the promise to regenerate the damaged periodontium. These cells have been identified within adult adipose tissue, periodontal ligament, and palatal tissue. Typical enzymatic isolation protocols are expensive, time consuming and often not clinically compliant. Enzyme-free, mechanical dissociation has been suggested as an alternative method of generating cell suspensions required for cell separation and subsequent expansion ex vivo. In our study, samples of rat skeletal muscle tissue were used to appraise the suitability of a novel mincing method of mechanical dissociation against enzymatic digestion with collagenase and dispase. Skeletal muscle is readily available and has been shown to contain NCSC populations. We used a Rigenera-Human Brain Wave® prototype mincer to produce a suspension of NCSCs. We have compared the resulting cell cultures produced via mechanical dissociation and enzymatic dissociation, producing single cell suspensions suitable for Magnetic Cell Sorting (MACs) and Fluorescence-activated cell sorting (FACS). Despite the Countess Automated Cytometry data demonstrating that cell suspensions produced by mechanical dissociation (n=24) contain on average 26.8 times as many viable cells as enzymatic cell suspensions (n=18), enzymatic suspensions produced more successful cell cultures. Spheroids and subsequently adherent cells formed from 4 enzymatic cell suspensions (44.4%) vs. 1 mechanical cell suspension (8.3%). Enzymatic digestion protocols formed spheroids faster and more plentifully than mechanical cell suspensions. Adherent cells and spheroids isolated via both methods appear morphologically similarly to NCSCs from our previous studies.

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Isolation of Human Male Germ-Line Stem Cells Using Enzymatic Digestion and Magnetic-Activated Cell Sorting

Methods in Molecular Biology - Germline Development ◽

10.1007/978-1-61779-436-0_4 ◽

2011 ◽

pp. 45-57 ◽

Cited By ~ 34

Author(s):

Zuping He ◽

Maria Kokkinaki ◽

Jiji Jiang ◽

Wenxian Zeng ◽

Ina Dobrinski ◽

...

Keyword(s):

Stem Cells ◽

Cell Sorting ◽

Germ Line ◽

Enzymatic Digestion ◽

Male Germ Line ◽

Human Male ◽

Magnetic Activated Cell Sorting

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138 IN VITRO CULTURE OF CD9-EXPRESSING CELLS ENRICHED BY MAGNETIC CELL SORTING FROM TESTES OF CRYPTORCHID ADULT AND PUP IN MICE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab138 ◽

2006 ◽

Vol 18 (2) ◽

pp. 177

Author(s):

T. Mitani ◽

Y. Tanaka ◽

Y. Ozaki ◽

K. Saeki ◽

K. Kato ◽

...

Keyword(s):

Stem Cells ◽

Cell Surface ◽

Germ Cells ◽

Cell Sorting ◽

Es Cells ◽

Immunohistochemical Analysis ◽

Cell Suspensions ◽

Magnetic Cell Sorting ◽

Fcm Analysis

Recently, studies on cell surface markers of spermatogonia in combination with germ cell transplantation techniques have made possible the functional analysis of germline stem cells (GS cells). The GS cells are downstream of the stem cells such as ES cells and embryonic germ cells (EG cells), which are derived from primordial germ cells (PGCs). Therefore, GS cells are expected to be useful in the production of genetically modified animals. In this study, we examined the enrichment and cultivation of mouse GS cells by magnetic cell sorting (MACS). Testicular cell suspensions were collected from C57BL/6J cryptorchid adult testes at 2 to 3 months after surgery and ICR pup (6 to 8 dpp) testes. They were digested by 0.1% collagenase followed by 0.25% trypsin with gentle shaking. Dissociated cell suspensions were filtrated through a glass-wool column followed by a Falcon cell strainer (40-�m mesh). They were then treated with biotin-conjugated anti-mouse CD9 antibody, whose antigen, CD9, is localized on the GS cell surface, followed by the streptavidin-microbeads treatment. The cell suspension was passed through a MACS-separation column. In Experiment I, MACS-treated fractions were analyzed by flow cytometry (FCM) on the rates of recovery and enrichment and their cellular characteristics. In Experiment II, CD9-positive (CD9+) cells were cultured on gelatin-coated MultiDish (176740, Nunc) with 4-5 � 105 cells/well in StemPro34-SFM supplemented with 1% fetal bovine serum, leukemia inhibitory factor, GDNF, bFGF, EGF, insulin, transferrin, putrescine, MEM vitamin solution, MEM-NEAA and some other reagents at 32�C or 37�C under 5% CO2 in air. They were examined for their proliferation and cytological changes such as CD9, �6-integrin and Oct-1 expression by immunohistochemistry. In Experiment I, MACS selection effectively enriched CD9+ cells from mouse testes. However, FCM analysis revealed that the CD9-negative (CD9-) cells partially remained in MACS-selected fraction from cryptorchid adult testes. In contrast, the CD9+ subpopulation could be successfully separated from CD9- subpopulation from pup testes. Therefore CD9+ subpopulation from pup testes was used for the following cultivation. In Experiment II, the cells proliferated in the first few days in suspension. Then they attached to the dish and formed colonies after 5 days or 3 days of culture at 32�C or 37�C, respectively. Immunohistochemical analysis showed that the cells maintained the expression of CD9 for at least 14 days, but their expression of �6-integrin gradually diminished. It was demonstrated by immunohistochemistry and FCM analysis that the cells in colonies expressed Oct-1, and its expression level was stronger in culture at 37�C than at 32�C. These findings indicate that the CD9+ cells collected from mouse pup testes have stem cell properties. This work was supported by the Wakayama Prefecture Collaboration of Regional Entities for the Advanced Technological Excellence, JST; by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan; and by a Grant-in Aid for Scientific Research from the Japan Society for the Promotion of Science.

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Methods of obtaining multipotent stem cells of the placenta in rats

Bulletin of Taras Shevchenko National University of Kyiv Series Biology ◽

10.17721/1728_2748.2015.70.73-77 ◽

2015 ◽

Vol 70 (2) ◽

pp. 73-77

Author(s):

G. Svitina ◽

L. Garmanchuk ◽

V. Shabliy

Keyword(s):

Stem Cells ◽

Cell Therapy ◽

Cell Cultures ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Enzymatic Digestion ◽

Optimal Method ◽

Rat Serum ◽

Multipotent Stem Cells ◽

Fetal Bovine

Placenta is a valuable source of multipotent stem cells (PMSC) widely used for cell therapy. Hence, the most optimal method of PMSC obtaining remains questionable. By methods of explant culturing and enzymatic digestion were obtained PMSC of fetal origin and multipotent features at 4 th passage. Allogeneic rat serum is not favor PMSC establishment and growth, consequently the use is made of fetal bovine serum, that is xenogeneic for cell cultures.

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Skeletal Muscle Regeneration: MSC versus Satellite Cells

Otolaryngology ◽

10.1016/j.otohns.2008.05.484 ◽

2008 ◽

Vol 139 (2_suppl) ◽

pp. P86-P86

Author(s):

Jens Stern-Straeter ◽

Juritz Stephanie ◽

Gregor Bran ◽

Frank Riedel ◽

Haneen Sadick ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Stem Cells ◽

Satellite Cell ◽

Cell Cultures ◽

Muscle Tissue ◽

Satellite Cells ◽

Myogenic Differentiation ◽

Culture Conditions ◽

Muscle Biopsies

Problem Differentiating stem cells into the myogenic linage in order to create functional muscle tissue is a challenging endeavour. In this work, adipose-derived mesenchymal stem cells (MSC) and satellite cells derived from muscle biopsies were compared regarding proliferation and myogenic differentiation potential under standardized cell culture conditions. This data was obtained in order to discover the most promising type of stem cell for regeneration of muscle tissue and to determine the optimal culture conditions for later clinical use. Methods Human MSC were isolated from adipose tissue, and primary human skeletal myoblasts were extracted from muscle biopsies by enzymatic digestion. Proliferation was analysed using the AlamarBlue® assay. Gene expression of marker genes – such as Myogenin, Myo D, Myf 5 and MHC – were analysed by RT-PCR. Immunostainings against desmin and sarcomeric-actin were performed as differentiation markers. Results MSC cell cultures showed a greater proliferation rate compared with satellite cell cultures. In both stem cell cultures, myogenic differentiation/heritage could be verified by immunostainings against the muscle-specific marker desmin. Gene expression and protein analysis revealed a more stable differentiation of human satellite cell cultures. Conclusion Characterization of both human MSC cultures and satellite cell cultures – and thereby an understanding of myogenesis – might lead to their clinical usage in skeletal muscle tissue engineering. The results in this study appear to indicate that human satellite cell cultures have a more stable differentiation under in vitro conditions and that they might offer a greater potential for skeletal muscle tissue engineering purposes. Significance Our study contributes to the understanding of myogenic differentiation of MSC and satellite cells and helps to improve culture systems for later clinical utilization.

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