Oct4 and nanog directly regulate dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells

The roles of Oct4 and Nanog in maintaining self-renewal and undifferentiated status of adult stem cells are unclear. Here, increase in Oct4 and Nanog expression along with increased proliferation and differentiation potential but decreased spontaneous differentiation were observed in early-passage (E), hypoxic culture (H), and p21 knockdown (p21KD) mesenchymal stem cells (MSCs) compared to late-passage (L), normoxic culture (N), and scrambled shRNA-overexpressed (Scr) MSCs. Knockdown of Oct4 and Nanog in E, H, and p21KD MSCs decreased?proliferation and differentiation potential and enhanced spontaneous differentiation, whereas overexpression of Oct4 and Nanog in L, N, and Scr MSCs increased proliferation and differentiation potential and suppressed spontaneous differentiation. Oct4 and Nanog upregulate Dnmt1 through direct binding to its promoter, thereby leading to the repressed expression of p16 and p21 and genes associated with development and lineage differentiation. These data demonstrate the important roles of Oct4 and Nanog in maintaining MSC properties.     

Role of Nanog in the maintenance of marrow stromal stem cells during post natal bone regeneration

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ～50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ～80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ～3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ～50% decrease was seen in the expression of terminal osteogenic gene expression and a ～50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ～2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.     

Differential mesengenic potential and expression of stem cell-fate modulators in mesenchymal stromal cells from human-term placenta and bone marrow

Placenta has attracted increasing attention over the past decade as a stem cell source for regenerative medicine. In particular, the amniochorionic membrane has been shown to harbor populations of mesenchymal stromal cells (MSCs). In this study, we have characterized ex vivo expanded MSCs from the human amniotic (hAMSCs) and chorionic (hCMSCs) membranes of human full-term placentas and adult bone marrow (hBMSCs). Our results show that hAMSCs, hCMSCs, and hBMSCs express typical mesenchymal (CD73, CD90, CD105, CD44, CD146, CD166) and pluripotent (Oct-4, Sox2, Nanog, Lin28, and Klf4) markers but not hematopoietic markers (CD45, CD34). Ex vivo expanded hAMSCs were found to be of fetal origin, while hCMSCs cultures contained only maternal cells. Cell proliferation was significantly higher in hCMSCs, compared to hAMSCs and hBMSCs. Integrin profiling revealed marked differences in the expression of α subunits between the three cell sources. Cadherin receptors were consistently expressed on a subset of progenitors (ranging from 1% to 60%), while N-CAM (CD56) was only expressed in hAMSCs and hCMSCs but not in hBMSCs. When induced to differentiate, hAMSCs and hCMSCs displayed strong chondrogenic and osteogenic differentiation potential but very limited capacity for adipogenic conversion. In contrast, hBMSCs showed strong differentiation potential along the three lineages. These results illustrate how MSCs from different ontological sources display differential expression of cell-fate mediators and mesodermal differentiation capacity.     

Low intensity pulsed ultrasound (LIPUS) maintains osteogenic potency by the increased expression and stability of Nanog through spleen tyrosine kinase (Syk) activation

Mesenchymal stem cells (MSCs) are a powerful tool for cell-based, clinical therapies like bone regeneration. Therapeutic use of cell transplantation requires many cells, however, the expansion process needed to produce large quantities of cells reduces the differentiation potential of MSCs. Here, we examined the protective effects of low intensity pulsed ultrasound (LIPUS) on the maintenance of osteogenic potency. Primary osteoblastic cells were serially passaged between 2 and 12 times with daily LIPUS treatment. We found that LIPUS stimulation maintains osteogenic differentiation capacity in serially passaged cells, as characterized by improved matrix mineralization and Osteocalcin mRNA expression. Decreased expression of Nanog, Sox2, and Msx2, and increased expression of Pparg2 from serial passaging was recovered in LIPUS-stimulated cells. We found that LIPUS stimulation not only increased but also sustained expression of Nanog in primary osteoblasts and ST2 cells, a mouse mesenchymal stromal cell line. Nanog overexpression in serially passaged cells mimicked the recuperative effects of LIPUS on osteogenic potency, highlighting the important role of Nanog in LIPUS stimulation. Additionally, we found that spleen tyrosine kinase (Syk) is an important signaling molecule to induce Nanog expression in LIPUS-stimulated cells. Syk activation was regulated by both Rho-associated kinase 1 (ROCK1) and extracellular ATP in a paracrine manner. Interestingly, the LIPUS-induced increase in Nanog mRNA expression was regulated by ATP-P2X4-Syk Y323 activation, while the improvement of Nanog protein stability was controlled by the ROCK1-Syk Y525/526 pathway. Taken together, these results indicate that LIPUS stimulation recovers and maintains the osteogenic potency of serially passaged cells through a Syk-Nanog axis.     

Hypermethylation of microRNA-149 activates SDF-1/CXCR4 to promote osteogenic differentiation of mesenchymal stem cells

MicroRNAs (miRs) involve in osteogenic differentiation and osteogenic potential of mesenchymal stem cells (MSCs). Accordingly, the present study aimed to further uncover role miR-149 plays in osteogenic differentiation of MSCs with the involvement of the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) pathway. Initially, the osteogenic differentiation model was induced. Next, the positive expression of STRO-1 in periosteum, alkaline phosphatase (ALP) activity, osteocalcin (OCN) protein content, and the calcium deposition in MSCs were determined. MSCs were treated with DNA methyltransferase inhibitor 5-aza-CdR, SDF-1 neutralizing antibody, or CXCR4 antagonist AMD3100 to investigate their roles in osteogenic differentiation; with the expression of CD44, CD90, CD14, and CD45 detected. Furthermore, the levels of SDF-1 and CXCR4, and the genes related to stemness (Nanog, Oct-4, and Sox-2) were measured to explore the effects of miR-149. The obtained data revealed the upregulation of STRO-1 in the periosteum. miR-149 could specifically bind to SDF-1. Besides, increased miR-149 methylation, higher ALP activity and OCN content, decreased positive rates of CD44 and CD90, and increased positive rates of CD14 and CD45 were found in osteogenic differentiation of MSCs. Subsequently, 5-Aza-CdR treatment reversed the above-mentioned effects. MSCs were finally treated with SDF-1 neutralizing antibody or AMD3100 to decrease Nanog, Oct-4, and Sox-2 expression. Taken together these results, miR-149 hypermethylation has the potential to activate the SDF-1/CXCR4 pathway and further promote osteogenic differentiation of MSCs.     

In vitro effects of RU486 on proliferation and differentiation capabilities of human bone marrow mesenchymal stromal cells

Although exogenous glucocorticoids (GC) play a role in the regulation of bone marrow mesenchymal stem/stromal cells (MSCs) proliferation and differentiation, the function of endogenous GC is not well understood. The purpose of this study was to investigate the effect of the blockage of endogenous GC using RU486, an antagonist of the glucocorticoid receptor, on the in vitro proliferation and differentiation capabilities of human MSCs. We quantitatively measured cell proliferation of human MSCs after treatment with increasing concentrations of RU486. We also evaluated multiple MSC differentiation capabilities, as well as the expression of stemness and senescence genes after proliferation of these human cells in vitro in the presence of RU486 at 10(-8)M. It was observed that RU486 treatment significantly increases the proliferation of human MSCs, although the optimal dose of RU486 for this increase in proliferation differs depending on the gender of the MSC donor. This improvement in MSC proliferation with RU486 treatment was higher in MSCs from male donors than that from females. No effect of RU486 on MSC proliferation was observed in a steroid-free medium. RU486 pretreatment significantly increased the expression of mRNA for alkaline phosphatase in human MSCs and the mRNA expression of osteocalcin of these cells up-regulated earlier after their exposure to osteogenic differentiation medium. Although no statistical significance in terms of chondrogenic differentiation markers was detected, mRNA expression for aggrecan and collagen type 2 were higher in a majority of the RU486-pretreated donor MSCs than their untreated controls. No significant difference in terms of MSC adipogenic differentiation capabilities were observed after RU486 treatment. RU486 treatment up-regulated the expressions of FGF-2 and Sox-11 in human MSCs. These results indicate that blockage of endogenous GCs may be developed as a novel approach to effectively improve the proliferation and osteochondral differentiation capabilities of human MSCs for potential clinical applications. Additional studies will be required to determine the potential long-term effects of RU486 treatment on these bone marrow cells.     

Steroid regulation of proliferation and osteogenic differentiation of bone marrow stromal cells: A gender difference

Bone marrow mesenchymal stem cells (MSCs) are considered a potential cell source for stem cell-based bone tissue engineering. However, noticeable limitations of insufficient supply and reduction of differentiation potential impact the feasibility of their clinical application. This study investigated the in vitro function of steroids and gender differences on the proliferation and differentiation of rat MSCs. Bone marrow MSCs of age-matched rats were exposed to proliferation and osteogenic differentiation media supplements with various concentrations of 17β-estradiol (E2) and dexamethasone. Cell proliferation was measured by MTS assay; osteogenic markers and steroid-associated growth factors and receptors were evaluated by ELISA and real-time PCR. The results revealed that supplements of E2 and dexamethasone increase MSC proliferation in a biphasic manner. The optimal dose and interaction of steroids required to improve MSC proliferation effectively varied depending on the gender of donors. Supplementation of E2 effectively improves osteogenic differentiation markers including ALP, osteocalcin and calcium levels for MSCs isolated from both male and female donors. The mRNA of TGF-β1 and BMP-7 are also up-regulated. However, effective doses to maximally improve osteogenic potentials and growth factors for MSCs are different between male and female donors. The relationship between steroid receptors, osteogenic markers and cytokines are also varied by genders. The outcomes of the present study strongly indicate that steroids potentially function as an effective modulator to improve the capacity of MSCs in bone regeneration. It provides crucial information for improving and optimizing MSCs for future clinical application of bone regeneration.     

去卵巢骨质疏松山羊骨髓基质细胞成骨分化能力的研究

目的：研究在构建的去卵巢骨质疏松山羊动物模型中,骨髓基质细胞（MSCs）的生物学特性以及其成骨能力。方法：建立去卵巢骨质疏松山羊动物模型,使用全骨髓法获取去卵巢骨质疏松山羊（实验组）和正常山羊（对照组）MSCs,流式细胞仪检测实验组和对照组细胞周期及增殖指数（PI）;地塞米松诱导21d时油红O染色,观察成脂分化比例;成骨诱导液诱导14d,碱性磷酸酶（ALP）染色、检测ALP表达量。结果：对照组PI高于实验组;地塞米松诱导后实验组脂肪细胞比例明显高于对照组;成骨诱导第7d,对照组ALP的表达量明显高于实验组。结论：去卵巢骨质疏松山羊的MSCs增殖和成骨分化能力都降低,可能与骨质疏松症的发病机理有关。     

Antioxidant effects of chrysin-loaded electrospun nanofibrous mats on proliferation and stemness preservation of human adipose-derived stem cells

An ideal biomaterial in regenerative medicine should be able to regulate the stem cell proliferation without the loss of its pluripotency. Chrysin (Chr) is a naturally occurring flavone with a wide spectrum of biological functions including anti-inflammatory and anti-oxidant properties. The present study describes the influence of Chr-loaded nanofibrous mats on the regulation of proliferation and stemness preservation of adipose-derived stem cells (ADSCs). For this purpose, Chr-loaded poly (ε-caprolactone)/poly (ethylene glycol) (PCL/PEG) nanofibrous mats were produced via electrospinning process and the successful fabrication of these bioactive mats was confirmed by field emission scanning electron microscopy (FE-SEM) and fourier transform infrared spectroscopy. ADSCs were seeded on the nanofibers and their morphology, viability, and stemness expression were analyzed using FE-SEM, MTT, and qPCR assays after 2 weeks of incubation, respectively. The results display that ADSCs exhibit better adhesion and significantly increased viability on the Chr-loaded PCL/PEG nanofibrous mats in relative to the PCL/PEG nanofibers and tissue culture polystyrene. The greater viability of ADSCs on Chr based nanofibers was further confirmed by higher expression levels of stemness markers Sox-2, Nanog, Oct-4, and Rex-1. These findings demonstrate that Chr-loaded PCL/PEG electrospun nanofibrous mats can be applied to improve cell adhesion and proliferation while concurrently preserving the stemness of ADSCs, thus representing a hopeful potential for application in stem cell therapy strategies.     

Interleukin‐6 maintains bone marrow‐derived mesenchymal stem cell stemness by an ERK1/2‐dependent mechanism

Adult human mesenchymal stem cells (MSCs) hold promise for an increasing list of therapeutic uses due to their ease of isolation, expansion, and multi‐lineage differentiation potential. To maximize the clinical potential of MSCs, the underlying mechanisms by which MSC functionality is controlled must be understood. We have taken a deconstructive approach to understand the individual components in vitro, namely the role of candidate “stemness” genes. Our recent microarray gene expression profiling data suggest that interleukin‐6 (IL‐6) may contribute to the maintenance of MSCs in their undifferentiated state. In this study, we showed that IL‐6 gene expression is significantly higher in undifferentiated MSCs as compared to their chondrogenic, osteogenic, and adipogenic derivatives. Moreover, we found that MSCs secrete copious amounts of IL‐6 protein, which decreases dramatically during osteogenic differentiation. We further evaluated the role of IL‐6 for maintenance of MSC “stemness,” using a series of functional assays. The data showed that IL‐6 is both necessary and sufficient for enhanced MSC proliferation, protects MSCs from apoptosis, inhibits adipogenic and chondrogenic differentiation of MSCs, and increases the rate of in vitro wound healing of MSCs. We further identified ERK1/2 activation as the key pathway through which IL‐6 regulates both MSC proliferation and inhibition of differentiation. Taken together, these findings show for the first time that IL‐6 maintains the proliferative and undifferentiated state of bone marrow‐derived MSCs, an important parameter for the optimization of both in vitro and in vivo manipulation of MSCs. J. Cell. Biochem. 108: 577–588, 2009. Published 2009 Wiley‐Liss, Inc.     

Caveolin‐1 regulates proliferation and osteogenic differentiation of human mesenchymal stem cells

Caveolin‐1 is a scaffolding protein of cholesterol‐rich caveolae lipid rafts in the plasma membrane. In addition to regulating cholesterol transport, caveolin‐1 has the ability to bind a diverse array of cell signaling molecules and regulate cell signal transduction in caveolae. Currently, there is little known about the role of caveolin‐1 in stem cells. It has been reported that the caveolin‐1 null mouse has an expanded population of cells expressing stem cell markers in the gut, mammary gland, and brain, suggestive of a role for caveolin‐1 in stem cell regulation. The caveolin‐1 null mouse also has increased bone mass and an increased bone formation rate, and its bone marrow‐derived mesenchymal stem cells (MSCs) have enhanced osteogenic potential. However, the role of caveolin‐1 in human MSC osteogenic differentiation remains unexplored. In this study, we have characterized the expression of caveolin‐1 in human bone marrow derived MSCs. We show that caveolin‐1 protein is enriched in density gradient‐fractionated MSC plasma membrane, consisting of ～100 nm diameter membrane‐bound vesicles, and is distributed in a punctate pattern by immunofluoresence localization. Expression of caveolin‐1 increases in MSCs induced to undergo osteogenic differentiation, and siRNA‐mediated knockdown of caveolin‐1 expression enhances MSC proliferation and osteogenic differentiation. Taken together, these findings suggest that caveolin‐1 normally acts to regulate the differentiation and renewal of MSCs, and increased caveolin‐1 expression during MSC osteogenesis likely acts as a negative feedback to stabilize the cell phenotype. J. Cell. Biochem. 113: 3773–3787, 2012. © 2012 Wiley Periodicals, Inc.     

miR-146a-5p circuitry uncouples cell proliferation and migration,but not differentiation,in human mesenchymal stem cells

Administration of mesenchymal stem cells (MSCs) has the potential to ameliorate degenerative disorders and to repair damaged tissues. The homing of transplanted MSCs to injured sites is a critical property of engraftment. Our aim was to identify microRNAs involved in controlling MSC proliferation and migration. MSCs can be isolated from bone marrow and umbilical cord Wharton’s jelly (BM-MSCs and WJ-MSCs, respectively), and WJ-MSCs show poorer motility yet have a better amplification rate compared with BM-MSCs. Small RNA sequencing revealed that miR-146a-5p is significantly overexpressed and has high abundance in WJ-MSCs. Knockdown of miR-146a-5p in WJ-MSCs inhibited their proliferation yet enhanced their migration, whereas overexpression of miR-146a-5p in BM-MSCs did not influence their osteogenic and adipogenic potentials. Chemokine (C-X-C motif) ligand 12 (CXCL12), together with SIKE1, which is an I-kappa-B kinase epsilon (IKKε) suppressor, is a direct target of miR-146a-5p in MSCs. Knockdown of miR-146a-5p resulted in the down-regulation of nuclear factor kappa-B (NF-κB) activity, which is highly activated in WJ-MSCs and is known to activate miR-146a-5p promoter. miR-146a-5p is also downstream of CXCL12, and a negative feedback loop is therefore formed in MSCs. These findings suggest that miR-146a-5p is critical to the uncoupling of motility and proliferation of MSCs. Our miRNome data also provide a roadmap for further understanding MSC biology.     

Moderate Exercise Mitigates the Detrimental Effects of Aging on Tendon Stem Cells

Aging is known to cause tendon degeneration whereas moderate exercise imparts beneficial effects on tendons. Since stem cells play a vital role in maintaining tissue integrity, in this study we aimed to define the effects of aging and moderate exercise on tendon stem/progenitor cells (TSCs) using in vitro and in vivo models. TSCs derived from aging mice (9 and 24 months) proliferated significantly slower than TSCs obtained from young mice (2.5 and 5 months). In addition, expression of the stem cell markers Oct-4, nucleostemin (NS), Sca-1 and SSEA-1 in TSCs decreased in an age-dependent manner. Interestingly, moderate mechanical stretching (4%) of aging TSCs in vitro significantly increased the expression of the stem cell marker, NS, but 8% stretching decreased NS expression. Similarly, 4% mechanical stretching increased the expression of Nanog, another stem cell marker, and the tenocyte-related genes, collagen I and tenomodulin. However, 8% stretching increased expression of the non-tenocyte-related genes, LPL, Sox-9 and Runx-2, while 4% stretching had minimal effects on the expression of these genes. In the in vivo study, moderate treadmill running (MTR) of aging mice (9 months) resulted in the increased proliferation rate of aging TSCs in culture, decreased lipid deposition, proteoglycan accumulation and calcification, and increased the expression of NS in the patellar tendons. These findings indicate that while aging impairs the proliferative ability of TSCs and reduces their stemness, moderate exercise can mitigate the deleterious effects of aging on TSCs and therefore may be responsible for decreased aging-induced tendon degeneration.     

Gene Expression Profiles of Human Mesenchymal Stromal Cells Derived from Wharton’s Jelly and Amniotic Membrane before and after Osteo-Induction Using NanoString Platform

The use of perinatal mesenchymal stem cells (MSCs) in bone tissue regeneration and engineering to substitute bone marrow MSCs has drawn great interest due to their high yield, ease of procurement, multilineage differentiation potential and lack of ethical concerns. Although amniotic membrane (AM) and Wharton’s jelly (WJ)-derived MSCs have been widely shown to possess osteogenic differentiation potential, the intrinsic properties determining their osteogenic capacity remain unclear. Here, we compared gene expression profiles of AM- and WJ-MSCs at basal and osteogenic conditions by using the NanoString Stem Cell Panel containing regulatory genes associated with stemness, self-renewal, Wnt, Notch and Hedgehog signalling pathways. At basal condition, WJ-MSCs displayed higher expression in most genes regardless of their functional roles in self-renewal, adhesion, or differentiation signalling pathways. After osteo-induction, elevated expression of self-renewal genes ADAR and PAFAH1B1 was observed in AM-MSCs, while stemness genes MME and ALDH1A1 were upregulated in WJ-MSC. Both MSCs showed differences in genes associated with ligands, receptors and ubiquitin ligases of the Notch pathway. In addition, further evidence was demonstrated in some signalling molecules including CTBPs, protein kinases, phosphatases, RHOA, RAC1. Downstream targets HES1 and JUN especially showed higher expression in non-induced WJ-MSCs. Hedgehog genes initially expressed in both MSCs were downregulated in WJ-MSCs during osteogenesis. This study has provided insights into the intrinsic biological differences that may lead to their discrimination in therapeutic intervention.     

Evidence of specific characteristics and osteogenic potentiality in bone cells from tibia

Human bone cells used for in vitro studies are mainly derived from bone marrow (BM) or trabecular bone (TB). There are no specific markers or procedures for isolation and growth of these cells. To validate the potentiality of these cells, we isolated human mesenchymal stromal cells (MSCs) and osteoblasts (OBs) from the tibial plateau of the same subject, grown in two different media (α-MEM and DMEM/F12) and analyzed for cell growth, proliferation, phenotype and osteogenic potential. We found that OBs grew well in both media tested, but MSCs were able to grow only in α-MEM medium. OBs in DMEM/F12 showed reduced proliferation capability and expressed a low level of alkaline phosphatase (AP), RUNX-2, osteocalcin (OC), bone sialoprotein (BSP), collagen type I (Col.I) compared with OBs in α-MEM but high level of collagen type XV (Col.XV). Compared with MSCs in α-MEM, OBs have an increased ability to proliferate and express more OC and BSP at molecular level but less AP, RUNX-2 and Col.I than MSCs. Time-course experiments to analyze the osteogenic potential of these cells showed that OBs were more efficient than MSCs. However, these cells obtained from tibial plateau showed a different trend of AP, OC and Col.I osteogenic markers compared to control MSCs from the iliac crest. This study shows that bone-adherent OBs grown in α-MEM medium are more efficient for osteogenic differentiation than BM MSCs and contribute to defining their phenotypic and functional characteristics, so providing a rationale for their use in bone tissue engineering or therapeutic purposes.     

Osteo-regenerative potential of ovarian granulosa cells: an in vitro and in vivo study

Granulosa cells (GC) express stemness markers and can differentiate into cell types not present within the follicles. Given that follicles at different stages of development populate the ovary, we undertook this research in the pig model to identify the stage of follicle, growing or luteinizing, from which GC with the best regenerative potential can be retrieved. Growing follicles were isolated from prepubertal gilts 50 h after equine chorionic gonadotropin (eCG) (1,200 IU) administration. Luteinizing follicles were obtained from prepubertal gilts treated with eCG (1,200 IU) followed, 60 h later, by hCG (500 IU). The follicles were isolated 30 h after hCG. The GC isolated from growing (GGC) and from luteinizing (LGC) follicles were expanded in vitro for three passages and exposed to osteogenic medium to trigger differentiation. The GC incorporated in PLGA scaffolds were cultured in osteogenic medium for 2 wks and then implanted subcutaneously in the dorsal region of SCID mice to assess their osteogenic potential in vivo. In addition to the typical granulosa cells characteristics (inhibin, progesterone and estrogen production and FSH receptors), GGC and LGC showed a diffused expression of the stemness markers Sox2, Nanog and TERT immediately after isolation. Expansion caused in both cell types a rapid disappearance of granulosa cell characters while it did not modify stemness marker expression. Osteogenic medium induced a marked extracellular matrix mineralization and alkaline phosphatase activation in LGC, clearly detectable after two wks, while the process was much lighter in GGC, where it became evident after 3 wks. Osteocalcin and Runx2 expressions were upregulated and stemness markers downregulated by osteogenic medium. The GC loaded implants, retrieved 8 wks after transplantation, had viable GC surrounding the several nodules of calcifications recorded. Similar effects were induced by GGC and LGC while calcification nodules were not recorded when scaffolds without cells were implanted. These data confirm that GC, expanded in vitro undergo progressive de-differentiation retaining their plasticity and demonstrate that both GGC and LGC have osteogenic potential, luteinizing cells being more efficient. Transplanted in SCID mice, GC participate in new bone formation, thus confirming their therapeutic potential.