In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

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Characterisation of the deleted in azoospermia like (Dazl)–green fluorescent protein mouse model generated by a two-step embryonic stem cell-based strategy to identify pluripotent and germ cells

Reproduction Fertility and Development ◽

10.1071/rd14253 ◽

2016 ◽

Vol 28 (11) ◽

pp. 1741 ◽

Cited By ~ 2

Author(s):

Priscila Ramos-Ibeas ◽

Eva Pericuesta ◽

Raúl Fernández-González ◽

Alfonso Gutiérrez-Adán ◽

Miguel Ángel Ramírez

Keyword(s):

Stem Cells ◽

Green Fluorescent Protein ◽

Transgenic Mice ◽

Germ Cells ◽

Fluorescent Protein ◽

Preimplantation Embryo ◽

Embryonic Stem ◽

Deleted In Azoospermia ◽

Green Fluorescent

The deleted in azoospermia like (Dazl) gene is preferentially expressed in germ cells; however, recent studies indicate that it may have pluripotency-related functions. We generated Dazl–green fluorescent protein (GFP) transgenic mice and assayed the ability of Dazl-driven GFP to mark preimplantation embryo development, fetal, neonatal and adult tissues, and in vitro differentiation from embryonic stem cells (ESCs) to embryoid bodies (EBs) and to primordial germ cell (PGC)-like cells. The Dazl-GFP mice were generated by a two-step ESC-based strategy, which enabled primary and secondary screening of stably transfected clones before embryo injection. During preimplantation embryo stages, GFP was detected from the zygote to blastocyst stage. At Embryonic Day (E) 12.5, GFP was expressed in gonadal ridges and in neonatal gonads of both sexes. In adult mice, GFP expression was found during spermatogenesis from spermatogonia to elongating spermatids and in the cytoplasm of oocytes. However, GFP mRNA was also detected in other tissues harbouring multipotent cells, such as the intestine and bone marrow. Fluorescence was maintained along in vitro Dazl-GFP ESC differentiation to EBs, and in PGC-like cells. In addition to its largely known function in germ cell development, Dazl could have an additional role in pluripotency, supporting these transgenic mice as a valuable tool for the prospective identification of stem cells from several tissues.

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In Vitro Osteogenic Differentiation of Adipose Stem Cells After Lentiviral Transduction With Green Fluorescent Protein

Journal of Craniofacial Surgery ◽

10.1097/scs.0b013e3181bf04af ◽

2009 ◽

Vol 20 (6) ◽

pp. 2193-2199 ◽

Cited By ~ 18

Author(s):

Qian Wang ◽

Megan B. Steigelman ◽

John A. Walker ◽

Shuo Chen ◽

Peter J. Hornsby ◽

...

Keyword(s):

Stem Cells ◽

Green Fluorescent Protein ◽

Osteogenic Differentiation ◽

Fluorescent Protein ◽

Adipose Stem Cells ◽

Lentiviral Transduction ◽

Green Fluorescent

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Osteogenic Differentiation Potential of Human Bone Marrow and Amniotic Fluid-Derived Mesenchymal Stem Cells in Vitro & in Vivo

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2019.124 ◽

2019 ◽

Vol 7 (4) ◽

pp. 507-515 ◽

Cited By ~ 4

Author(s):

Eman E. A. Mohammed ◽

Mohamed El-Zawahry ◽

Abdel Razik H. Farrag ◽

Nahla N. Abdel Aziz ◽

Wessam Sharaf-ElDin ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Amniotic Fluid ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Differentiation Potential

BACKGROUND: Cell therapies offer a promising potential in promoting bone regeneration. Stem cell therapy presents attractive care modality in treating degenerative conditions or tissue injuries. The rationale behind this is both the expansion potential of stem cells into a large cell population size and its differentiation abilities into a wide variety of tissue types, when given the proper stimuli. A progenitor stem cell is a promising source of cell therapy in regenerative medicine and bone tissue engineering. AIM: This study aimed to compare the osteogenic differentiation and regenerative potentials of human mesenchymal stem cells derived from human bone marrow (hBM-MSCs) or amniotic fluid (hAF-MSCs), both in vitro and in vivo studies. SUBJECTS AND METHODS: Human MSCs, used in this study, were successfully isolated from two human sources; the bone marrow (BM) and amniotic fluid (AF) collected at the gestational ages of second or third trimesters. RESULTS: The stem cells derived from amniotic fluid seemed to be the most promising type of progenitor cells for clinical applications. In a pre-clinical experiment, attempting to explore the therapeutic application of MSCs in bone regeneration, Rat lumbar spines defects were surgically created and treated with undifferentiated and osteogenically differentiated MSCs, derived from BM and second trimester AF. Cells were loaded on gel-foam scaffolds, inserted and fixed in the area of the surgical defect. X-Ray radiography follows up, and histopathological analysis was done three-four months post- operation. The transplantation of AF-MSCs or BM-MSCs into induced bony defects showed promising results. The AF-MSCs are offering a better healing effect increasing the likelihood of achieving successful spinal fusion. Some bone changes were observed in rats transplanted with osteoblasts differentiated cells but not in rats transplanted with undifferentiated MSCs. Longer observational periods are required to evaluate a true bone formation. The findings of this study suggested that the different sources; hBM-MSCs or hAF-MSCs exhibited remarkably different signature regarding the cell morphology, proliferation capacity and osteogenic differentiation potential CONCLUSIONS: AF-MSCs have a better performance in vivo bone healing than that of BM-MSCs. Hence, AF derived MSCs is highly recommended as an alternative source to BM-MSCs in bone regeneration and spine fusion surgeries. Moreover, the usage of gel-foam as a scaffold proved as an efficient cell carrier that showed bio-compatibility with cells, bio-degradability and osteoinductivity in vivo.

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In Vivo and In Vitro Tissue-Specific Expression of Green Fluorescent Protein Using the Cre-Lox System in Mouse Embryonic Stem Cells

Stem Cells ◽

10.1634/stemcells.2004-0163 ◽

2005 ◽

Vol 23 (1) ◽

pp. 10-15 ◽

Cited By ~ 7

Author(s):

Jan Schindehütte ◽

Hidefumi Fukumitsu ◽

Patrick Collombat ◽

Gundula Griesel ◽

Christopher Brink ◽

...

Keyword(s):

Stem Cells ◽

Green Fluorescent Protein ◽

Embryonic Stem Cells ◽

Fluorescent Protein ◽

Embryonic Stem ◽

Specific Expression ◽

Tissue Specific Expression ◽

Green Fluorescent

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Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

Cell Transplantation ◽

10.1177/0963689720978219 ◽

2021 ◽

Vol 30 ◽

pp. 096368972097821

Author(s):

Andrea Tenorio-Mina ◽

Daniel Cortés ◽

Joel Esquivel-Estudillo ◽

Adolfo López-Ornelas ◽

Alejandro Cabrera-Wrooman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Ectopic Expression ◽

Neural Induction ◽

Human Keratinocytes ◽

Differentiation Potential ◽

Neuronal Proteins ◽

Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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