Density gradients for the isolation of germ cells from embryoid bodies

Saiti, Deshira; Lacham-Kaplan, Orly

doi:10.1016/s1472-6483(10)60489-0

Cited by 10 publications

(6 citation statements)

References 29 publications

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“…In an independent experiment we have shown Desmin to overlap with MYH expression in in-vitro derived C2C12 myotubes at 120 h of differentiation (Figure 1). For this, confluent cells (80-90%) were cultured in Con-Ve for 120 h. At 120 h, cultures were washed, fixed and analyzed by immunofluorescence as previously described [29,59]. Fixed cultures were stained with the appropriate secondary antibodies (Alexa Fluor 488; green and 594; red, Life Technologies) and costained with 4 ,6-diamidino-2-phenylindole (DAPI) (Life Technologies) following an overnight incubation with anti-mouse Desmin (Life Technologies) and anti-mouse skeletal muscle myosin heavy chain antibody (Life Technologies).…”

Section: Immunofluorescence Mytube Formation and Fusion Parametersmentioning

confidence: 99%

Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

Lacham-Kaplan

Camera

Hawley

2020

IJMS

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Estrogen (E2) and polyunsaturated fatty acids (n-3PUFA) supplements independently support general wellbeing and enhance muscle regeneration in-vivo and myotube formation in-vitro. However, the combined effect of E2 and n-3PUFA on myoblast differentiation is not known. The purpose of the study was to identify whether E2 and n-3PUFA possess a synergistic effect on in-vitro myogenesis. Mouse C2C12 myoblasts, a reliable model to reiterate myogenic events in-vitro, were treated with 10nM E2 and 50μM eicosapentaenoic acid (EPA) independently or combined, for 0–24 h or 0–120 h during differentiation. Immunofluorescence, targeted qPCR and next generation sequencing (NGS) were used to characterize morphological changes and differential expression of key genes involved in the regulation of myogenesis and muscle function pathways. E2 increased estrogen receptor α (Erα) and the expression of the mitogen-activated protein kinase 11 (Mapk11) within 1 h of treatment and improved myoblast differentiation and myotube formation. A significant reduction (p < 0.001) in myotube formation and in the expression of myogenic regulatory factors Mrfs (MyoD, Myog and Myh1) and the myoblast fusion related gene, Tmem8c, was observed in the presence of EPA and the combined E2/EPA treatment. Additionally, EPA treatment at 48 h of differentiation inhibited the majority of genes associated with the myogenic and striated muscle contraction pathways. In conclusion, EPA and E2 had no synergistic effect on myotube formation in-vitro. Independently, EPA inhibited myoblast differentiation and overrides the stimulatory effect of E2 when used in combination with E2.

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Section: Immunofluorescence Mytube Formation and Fusion Parametersmentioning

confidence: 99%

Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

Lacham-Kaplan

Camera

Hawley

2020

IJMS

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“…In one group of protocols, specific factors are added to the differentiating cells in order to increase germ cell derivation (BMP4 [5,[10][11][12], retinoic acid (RA) [7,[13][14][15], basic fibroblast growth factor (bFGF) [16], testicular cell conditioned medium [14,17], forskolin [15] and wingless-related MMTV integration site 3A (Wnt3a) [11]). Other protocols focus on the selection and enrichment of the spontaneously derived germ cell population [6,18,19].…”

Section: Introductionmentioning

confidence: 99%

Sertoli cell-conditioned medium induces germ cell differentiation in human embryonic stem cells

Geens

Sermon

Velde

et al. 2011

J Assist Reprod Genet

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Purpose To investigate the spontaneous germ cell differentiation capacity of VUB hESC lines, develop a protocol for the induction of germ cell differentiation using conditioned medium from Sertoli cells (SCCM) and compare it to existing protocols. Methods hESC were allowed to differentiate spontaneously or after the addition of bone morphogenetic proteins (BMPs) and/or SCCM. VASA transcripts were measured by relative quantification real-time RT-PCR to determine the efficiency of germ cell differentiation. Results VUB hESC lines can differentiate spontaneously towards the germ cell lineage, however, more consistently in an embryoid body approach than in monolayer cultures.BMPs and SCCM significantly improve VASA expression, but do not have a synergistic effect. Direct contact of differentiating hESC with Sertoli cells does not improve VASA expression. Conclusions SCCM contains inductive factors for germ cell differentiation and could represent an element for in-vitro differentiation to germ cells.

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“…Improvement of germ cell differentiation, evidenced by enhanced expression of pre-meiotic and meiotic germ cell-specific genes, was observed in iPSCs derived from embryoid bodies (EBs) ( 33 ). Selection of germ cells from these EBs may be achieved by a simple density gradient procedure ( 34 ). Furthermore, injection of testicular cells and iPSCs into the dorsal skin of mice led to reconstitution of seminiferous tubules, with iPSC-derived germ cells settling on basement membranes of reconstituted tubules ( 33 , 35 ).…”

Section: Methodsmentioning

confidence: 99%

Induced Pluripotent Stem Cell Potential in Medicine, Specifically Focused on Reproductive Medicine

Botman

Wyns

2014

Front. Surg.

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Since 2006, several laboratories have proved that somatic cells can be reprogramed into induced pluripotent stem cells (iPSCs). iPSCs have enormous potential in stem cell biology as they can give rise to numerous cell lineages, including the three germ layers. In this review, we discuss past and recent advances in human iPSCs used for modeling diseases in vitro, screening drugs to test new treatments, and autologous cell and tissue regenerative therapies, with a special focus on reproductive medicine applications. While this latter field of research is still in its infancy, it holds great promise for investigating germ cell development and studying the genetic and physiopathological mechanisms of infertility. A major cause of infertility is the absence of germ cells in the testes, mainly due to genetic background or as a consequence of gonadotoxic treatments. For these patients, no effective fertility restoration strategy has so far been identified. The derivation of germ cells from iPSCs represents an alternative source of stem cells able to differentiate into spermatozoa. Lessons learned from animal models as well as studies on human iPSCs for reproductive purposes are reviewed.

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Density gradients for the isolation of germ cells from embryoid bodies

Cited by 10 publications

References 29 publications

Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

Sertoli cell-conditioned medium induces germ cell differentiation in human embryonic stem cells

Induced Pluripotent Stem Cell Potential in Medicine, Specifically Focused on Reproductive Medicine

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