Saturday, October 10, 2009

Embryonic Stem Cells into Chondrocytes

In vitro differentiation and maturation of human embryonic stem cell into multipotent cells.

"endochondral ossification [has not been] obtained during in vitro culture of either MSCs or ESCs."  However, chondrocytes grown in vitro and then transferred in vivo have obtained endochondral ossification.

Chondrogenic differentiation of ESCs has been achieved with various combinations of IGF-1, BMP-2, TGFB1, Retinoic Acid, Ascorbic Acid, Dexamethasone, and TGFB3.

"constructs cultured in perfusion bioreactors showed significantly higher amounts of glycosaminoglycans (GAGs), DNA, total collagen, and collagen II in comparison to static culture. Mechanical stiffness of constructs increased 3.7-fold under dynamic culture conditions, and cells cultured in perfusion bioreactors highly expressed cartilage-related genes when compared with static culture."

Large-scale production of murine embryonic stem cell-derived osteoblasts and chondrocytes on microcarriers in serum-free media.

"There were two types of chondrogenic media which were used in separate cultures; (i) chondrogenic medium 1 (CM1) consisted of DMEM, supplemented with 15% Knockout-Serum Replacement, 1% non-essential amino acids, 1% penicillin/streptomycin, 1 mm Sodium Pyruvate, 100 mm β-mercaptoethanol, 50 μg/mL Ascorbic acid, 10 ng/mL BMP-2, 10 ng/mL TGFβ1 and 1% of an insulin–transferrin–selenium complex, and (ii) chondrogenic medium 2 (CM2) consisted of all of the components in CM1 minus the TGFβ1."

"For chondrogenic differentiation, maintenance medium was replaced with CM1 and bioreactors were seeded with day 6 cell-loaded microcarriers (CL + MCs) and agitated at 60 or 100 rpm. On day 5, CM1 was replaced with CM2 for the remainder of the differentiation. For all conditions, differentiation proceeded for a total of 30 days."

Improved media compositions for the differentiation of embryonic stem cells into osteoblasts and chondrocytes.

"a protocol for chondrogenic differentiation employs BMP-2 and transforming growth factor β1 as chondrogenic inducers, with 60% chondrogenic end point efficiency."

One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system.

" Mesoderm induction was achieved in cultures supplemented with BMP4, activin A, or Wnt3a. Prolonged culture with sustained activin A, TGFβ3, or BMP4 supplementation led to robust chondrogenic induction. A short pulse of activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with activin A or with activin A followed by TGFβ3 promoted articular cartilage formation. Thus, we devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting."

"greater Flk1{LSJL upregulates this as KDR} transcript expression was observed in BMP4- or Wnt3a-supplemented cultures than that in activin A-treated cultures"

" BMP4 was more inductive in the up-regulation of the posterior primitive streak markers Evx1, HoxB1{up in LSJL}, Tal1, and GATA2, while activin A appeared to be equally inductive in Lhx1. BMP4 was more effective in inducing the paraxial mesoderm marker PDGFRα than activin A, which up-regulated the anterior mesoderm marker Meox2{up in LSJL}, Fst, and Mesp2 to a lesser extent. Activin A effectively up-regulated the mesendoderm markers Gsc and Foxa2, and Wnt3a appeared to have a pan-mesodermal inductive effect."

"both FGF8[FGF8 causes poor matrix attachment]- and TGFβ3-treated cultures also possessed Sox9-expressing populations"

"ESCs were differentiated into the chondrocyte lineage, forming small cartilaginous aggregates in suspension. Differentiated ESCs showed that chondrogenesis was typically characterized by five overlapping stages. During the first stage, cell condensation and aggregate formation was observed. The second stage was characterized by differentiation into chondrocytes and fibril scaffold formation within spherical aggregates. Deposition of cartilaginous extracellular matrix and cartilage formation were hallmarks of the third stage. Apoptosis of chondrocytes, hypertrophy and/or degradation of cartilage occurred during the fourth stage. Finally, during the fifth stage, bone replacement with membranous calcified tissues took place."

"The human ESCs were cultured as an aggregate in a pellet culture system for 14 days in basal chondrogenic medium (CM), CM with TGFbeta1, CM with BMP-7, or CM with both TGFbeta1 and BMP-7.
The size and wet weight of the cartilage pellets and glycosaminoglycan levels increased, with the smallest, intermediate, and greatest increases, respectively, observed with CM plus TGFbeta1 treatment, CM plus BMP-7 treatment, and CM plus TGFbeta1 and BMP-7 treatment (compared with CM treatment alone). The largest size and highest weight of the pellet was in the group in which TGFbeta1 and BMP-7 were added to the medium. However, expression of the genes for cartilage-specific aggrecan and type II collagen II, as assessed by determination of messenger RNA levels, was highest in the BMP-7-treated group. Superficial zone protein (SZP)/lubricin, a marker of the superficial zone articular chondrocyte, was not detectable under identical culture conditions."

The effects of low oxygen on self-renewal and differentiation of embryonic stem cells.

Differentiation under low oxygen increases number of chondrocytes due to HIF1a which degrades under high oxygen conditions.

"we focused on the in vitro chondrocyte differentiation of ESCs through micro-mass without using an embryoid body (EB) step and observed the unique characteristics of cartilage formation coupled with endochondral ossification in vivo. This approach resulted in an aggressive loss of discordant cells by apoptosis, which was accompanied by significant changes in gene expression during the course of ESC differentiation into chondrocytes. Unlike EB formation where discordant cells remain trapped within aggregates, micro-mass permits cells to die, leave the group and/or form a new group in response to changes in gene expression. Our observations suggest that the cell death that accompanies ESC micro-mass differentiation helps purify a terminally differentiated cell population and selects for targeted end points within a suitable microenvironment."

"Whereas mouse ESCs show consistent cartilage formation, we did not observe cartilage formation when the chondrogenic protocols were transferred to human ESCs. Not only did the serum-free chondrogenic medium containing TGFβ3 result in heterogeneous cartilage formation by mouse ESCs, but also the cartilaginous tissue that was formed by human MSCs in our studies was not homogeneous, even when supplemented with BMP6."

Chondrogenic derivatives of embryonic stem cells seeded into 3D polycaprolactone scaffolds generated cartilage tissue in vivo.

"ESCs were induced to differentiate into chondroprogenitors, capable of proliferating and subsequently differentiating into cartilage-producing cells. The chondrogenic cells expressed chondrocyte-specific markers and deposited extracellular matrix proteins, proteoglycans. ESC-derived chondrogenic cells and polycaprolactone scaffolds seeded with these cells implanted in mice (129 SvImJ) generated cartilage tissue in vivo. Postimplant analysis of the retrieved tissues demonstrated cartilage-like tissue formation in 3-4 weeks. The cells of retrieved tissues also expressed the chondrocyte-specific marker collagen II. These findings suggest that ESCs can be used for tissue engineering and cultivation of cartilage tissues."

"galectin-3 expression is predominantly found in the early hypertrophic chondrocytes of the growth plate, but not in the terminally mature chondrocytes."

Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment.

"Co-cultures of hESC and chondrocytes were established using well inserts, with control comprising hESC grown alone or with fibroblasts. After 28 days, after removal of the chondrocyte inserts, hESC differentiation was assessed, by morphology, immunocytochemistry, and reverse transcription polymerase chain reaction. hESC, co-cultured or grown alone, were also implanted into SCID mice on a poly-D, L-lactide scaffold, harvested 35 days later and assessed in the same way. hESC co-cultured with chondrocytes formed colonies and secreted extracellular matrix containing glycosaminoglycans (GAG). Quantitative assay showed increased synthesis of sulfated GAG in co-culture as compared with control hESC grown alone for the same period (p < 0.0001). In addition, co-cultured hESC expressed Sox 9 and collagen type II, unlike control hESC. Co-culture with fibroblasts did not induce chondrogenic differentiation. The implanted constructs with co-cultured hESC contained significantly more type II collagen (p < 0.01), type I collagen (p < 0.05), total collagen (p < 0.01), and GAG (p < 0.01) than those with hESC grown alone. Thus, we show for the first time differentiation of hESC to chondrocytes."

"Control hESC were found to form some colonies after 28 days’ culture alone but did not express any refractile matrix"

Epigenetic modification of retinoic acid-treated human embryonic stem cells.

"Epigenetic modification of the genome through DNA methylation is the key to maintaining the differentiated state of human embryonic stem cells (hESCs), and it must be reset during differentiation by retinoic acid (RA) treatment. A genome-wide methylation/gene expression assay was performed in order to identify epigenetic modifications of RA-treated hESCs. Between undifferentiated and RA-treated hESCs, 166 differentially methylated CpG sites and 2,013 differentially expressed genes were discovered. Combined analysis of methylation and expression data revealed that 19 genes (STAP2, VAMP8, C10orf26, WFIKKN1, ELF3, C1QTNF6, C10orf10, MRGPRF{up in LSJL}, ARSE, LSAMP, CENTD3, LDB2, POU5F1, GSPT2, THY1, ZNF574, MSX1, SCMH1, and RARB) were highly correlated with each other."

A human embryonic stem cell-derived clonal progenitor cell line with chondrogenic potential and markers of craniofacial mesenchyme.

"The 4D20.8 cell line was compared with mesenchymal stem cells and dental pulp stem cells by assessing osteochondral markers using immunohistochemical methods, gene expression microarrays, quantitative real-time PCR and in vivo repair of rat articular condyles.
4D20.8 expressed the site-specific gene markers LHX8{up in LSJL} and BARX1 and robustly upregulated chondrocyte markers upon differentiation. Differentiated 4D20.8 cells expressed relatively low levels of COL10A1 and lacked IHH and CD74 expression. Transplantation of 4D20.8 cells into experimentally induced defects in the femoral condyle of athymic rats resulted in cartilage and bone differentiation approximating that of the original tissue architecture. Relatively high COL2A1 and minimal COL10A1 expression occurred during differentiation in HyStem-C hydrogel with TGF-β3 and GDF-5."

Embryonic Stem Cells Incorporate into Newly Formed Bone and do not Form Tumors in an Immunocompetent Mouse Fracture Model.

"Male ES cells were differentiated either into osteoblasts or chondrocytes. The differentiated cells were implanted into a burr-hole fracture created in the proximal tibiae of immunocompetent female mice, strain-matched to the ES cell line. The ability of the differentiated ES cell-derived cells (bearing the Y chromosome) to incorporate into the newly formed bone was assessed. ES cells differentiated with either osteogenic or chondrogenic medium supplementation formed a soft-tissue mass that disrupted the normal bone architecture by 4 weeks after implantation in some mice. In contrast, mice receiving osteoblastic cells that were differentiated in a 3-dimensional type I collagen gel showed evidence of new bone formation at the defect site without evidence of tumor formation for up to 8 weeks after implantation. In this injury model, type I collagen is more effective than medium supplementation at driving more complete differentiation of ES cells, as evidenced by reducing their tumorigenicity."

"ES cells exposed to chondrogenic medium supplements (CHON medium) displayed a significant increase in glycosaminoglycan production, positive mRNA and protein expression of collagen II and aggrecan, and formed cartilaginous nodules following subcutaneous implantation of the cells into immunecompromised mice"

"pluripotent murine D3 ES cell line from male Sv129 mice"  8 week old mice were used for the recipients.

Chondrogenic medium consisted of ITS, FBS, TGF-B1, BMP-2, and ascorbic acid.

The fracture was created by "a small incision  on the medial side of the left proximal tibia and a 0.7mm hole was drilled though the medial cortex and though the medullary cavity of the metaphysis using a high-speed microdrill"

"all burr-holes were located distal to the growth plate, and had disrupted one side of the cortical bone as well as the trabecular bone in the medullary cavity"

"The mean burr-hole diameter was 0.71±0.04 mm, the mean distance from the centre of the hole to the top of the epiphysis was 2.49±0.26 mm and the mean depth of the hole was 1.07±0.21 mm"

"In the non-treated fracture control group (CTL), trabecular bone bridged the cortical bone defect and filled the medullary cavity at the injury site by 2 weeks. By 8 weeks, the trabecular bone volume in the medullary cavity was similar to that observed in the non-injured (NI) bones, and the cortical defect site was bridged with compact bone"

"By 4 weeks post-implantation, 2 of the 3 mice that had received medium-induced chondrogenic cells (CHON)  had abnormal healing of the cortex at the burr-hole site. Specifically, a large cavity was found in the medial cortex. By 8 weeks post-implantation, 1 of 3 CHON mice had severe disruption of the bone tissue architecture throughout the proximal tibia. The presence of a soft tissue mass containing cysts surrounded by striated muscle tissue was observed at the fracture site. Further, by 8 weeks post-implantation in the CHON treated bone, two to three layers of lamellar bone had formed along borders where the host bone met with the tumor, indicative of reactive bone formation. Normal bone healing
was observed in the other CHON treated mice."

Enhancement of osteogenic and chondrogenic differentiation of human embryonic stem cells by mesodermal lineage induction with BMP-4 and FGF2 treatment.

"Bone morphogenetic protein 4 (BMP4) alone or BMP4 combined with fibroblast growth factor 2 (FGF2) treatment enhanced mesodermal differentiation of human embryonic stem cells (hESCs) that were cultured feeder-free on Matrigel. Mesodermal lineage-induced embryoid bodies (EBs) generate greater numbers of osteogenic and chondrogenic lineage cells. To induce the mesodermal lineage, hESCs were treated with BMP4 and FGF2 during the EB state. The treatment decreased endodermal and ectodermal lineage gene expression and increased mesodermal lineage gene expression. The mesodermal lineage-induced EBs underwent enhanced osteogenic and chondrogenic differentiation after differentiation induction. This method could enhance [the] chondrogenic differentiation of hESCs{specifically expression of Type II Collagen and Sox9 was enhanced}."

Combined BMP4 and FGF2 treatment increased mesendermal gene WNT3 and decreased other lineage genes Zic, Pax6, Sox1, and Foxq1.

"BMP4 signaling is needed for mesoderm formation"

Heterogeneity of Embryonic and Adult Stem Cells

"stem cells of embryonic, neural, and hematopoietic origin are heterogeneous, with cells moving between two or more metastable states. These cell states show a bias in their differentiation potential and correlate with specific patterns of transcription factor expression and chromatin modifications."

"approximately 80% of ESCs express Nanog, while 10%–20% do not. In addition, Gata6, a transcription factor governing primitive endoderm formation[cartilage is mesoderm derived lineasge], is predominantly expressed in Nanogneg cells. ESCs also display heterogeneity with regard to expression of the transcription factor Rex1"

"Stella, also known as PGC7 or Dppa3{up in LSJL}, has been implicated in the maintenance of gene-specific DNA methylation in the early embryo"

"H3K4me3 and H3K9ac, histone modifications associated with gene activation, were more prevalent in Stella-GFPpos than in Stella-GFPneg ESCs and lowest in EpiSCs."

"HSCs fall into two (or more) subpopulations that exhibit distinct self-renewal and differentiation biases {but not to chondrocytes}"

"The effects of Notch are mediated by upregulation of the helix-loop-helix transcription factor (HLH) Hes1{up}."

"Hes1 oscillations drive Delta-like1 and Neurogenin2 oscillations. Notch signaling is required for the induction of Hes1 oscillations under physiological conditions."

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates.

"The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs).  The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSC. In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cases and, in some conditions, markers of tendon differentiation."

I can't get this full study :(

Specification of chondrocytes and cartilage tissues from embryonic stem cells.

"mouse ESC-derived chondrogenic mesoderm arises from a Flk-1−/Pdgfrα+ (F−P+) population that emerges in a defined temporal pattern following the development of an early cardiogenic F−P+ population. Specification of the late-arising F−P+ population with BMP4 generated a highly enriched population of chondrocytes expressing genes associated with growth plate hypertrophic chondrocytes. By contrast, specification with Gdf5, together with inhibition of hedgehog and BMP signaling pathways, generated a population of non-hypertrophic chondrocytes that displayed properties of articular chondrocytes. The two chondrocyte populations retained their hypertrophic and non-hypertrophic properties when induced to generate spatially organized proteoglycan-rich cartilage-like tissue in vitro. Transplantation of either type of chondrocyte, or tissue generated from them, into immunodeficient recipients resulted in the development of cartilage tissue and bone within an 8-week period. Significant ossification was not observed when the tissue was transplanted into osteoblast-depleted mice or into diffusion chambers that prevent vascularization. Thus, through stage-specific manipulation of appropriate signaling pathways it is possible to efficiently and reproducibly derive hypertrophic and non-hypertrophic chondrocyte populations from mouse ESCs that are able to generate distinct cartilage-like tissue in vitro and maintain a cartilage tissue phenotype within an avascular and/or osteoblast-free niche in vivo. "

"Chondrocytes in the vertebrae and ribs develop from paraxial mesoderm, whereas chondrocytes in the long bones and most of the girdles are derived from lateral plate mesoderm (LPM), which also gives rise to hematopoietic and cardiovascular lineages "

"Following induction, strips of paraxial mesoderm are segmented into somites. Somite development is regulated, in part, by the transcription factors paraxis (Tcf15) and Tbx18, the expression of which coincides with the induction of paraxial mesoderm. Individual somites are then patterned into the ventral sclerotome, which forms the axial skeleton, including cartilage and the vertebral column, and the dorsal dermomyotome which develops into skeletal muscles and the dermis of the back. Specification of the sclerotome is marked by the expression of two transcription factors, Meox1 and Nkx3.2 (also known as Bapx1). A population of collagen 2 (Col2a1)-positive mesenchymal cells with chondrogenic potential develops from sclerotome-derived cells at embryonic day (E) 12.5 of mouse development"

" Specification of this chondrogenic mesoderm with either BMP4 or the combination of Gdf5, cyclopamine and soluble Bmpr1α (sBmpr1α) resulted in the development of populations with hypertrophic and non-hypertrophic chondrocyte characteristics, respectively."<-So BMP4 could be useful for osteochondral induction.

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