Friday, October 16, 2009

iPSCs to chondrocytes

iPSCs were also discussed here.

Chondrogenic Differentiation in vitro of Murine Two-Factor Induced Pluripotent Stem Cells is Comparable to Murine Embryonic Stem Cells.

"Differentiation of embryonic stem (ES) cells via embryoid bodies has been established as an appropriate model to study the development of various cell types in vitro. Here, we show that murine induced pluripotent stem (iPS) cells, reprogrammed by exogenous expression of the two transcription factors Oct4 and Klf4 (2F OK iPS), differentiate into chondrocytes in vitro characterized by the appearance of Alcian blue-stained nodules and the expression of cartilage-associated genes and proteins. Quantitatively, the chondrogenic differentiation potential of 2F OK iPS and ES cells was found to be similar. Further, we demonstrate the induction of chondrogenic iPS cell differentiation by certain members of the transforming growth factor-β family (BMP-2, TGF-β(1)). The number of Alcian blue-positive nodules and the expression of the cartilage marker molecule collagen type II increased after application of BMP-2, whereas simultaneous treatment with both BMP-2 and TGF-β(1) showed no significant effect on gene expression."

"Oct4 together with Klf4 is sufficient to reprogram neural stem cells into pluripotent stem cells, so-called two-factor (2F) OK iPS cells"

"Growing on mitotically inactivated MEF undifferentiated murine iPS cells of line 2F OK, clone F-4, (2F OK iPS) formed colonies very similar to ES cells of line D3 (ES1) but smaller in size"

"Examination of the proliferation rate of 2F OK iPS cells with ES1 cells demonstrated that the analyzed ES cell line proliferated approximately 1.9-fold faster than the iPS cells. At the time of assessment, 40% of the iPS cells had entered the S-phase of the cell cycle, in contrast to 74% in the case of the ES cells"

iPS had lower Nanog expression but higher Oct4 expression.

"subtle differences observed between the analyzed iPS and ES cells may be due to clonal variation."

"2F OK iPS cell-derived EBs expressed somewhat higher levels of collagen type II compared to ES1 cell-derived EBs."

"At 5 + 14 days, BMP-2-treated 2F OK iPS cell-derived EBs expressed about 2.6-fold higher levels of collagen type II than the control EBs."

"Treatment of 2F OK iPS cell-derived EBs with 10 ng/ml BMP-2 and 2 ng/ml TGF-β1 during the suspension phase (2–5 days) resulted in an even higher and statistically significant increase in Alcian blue-positive nodules"

"the induction of chondrogenesis by simultaneous application of BMP-2 and TGF-β1 could not be confirmed by real-time PCR analysis"

"Various iPS cell lines generated from different cell types in fact show remarkable differences with respect to pluripotency marker expression. In general, differences among iPS cells and between ES and iPS cells have been reported which are most probably caused by epigenetic marks of the cell type of origin"

"murine iPS cells can be differentiated into various cell types using the same protocols used for ES cells but often less efficiently compared to ES cells"

"iPS cells retain an epigenetic memory of their tissue of origin that might hinder or even limit their differentiation propensity, favoring differentiation along lineages not related to the donor cell type"

[Direct cell reprogramming to chondrogenic cells from dermal fibroblast culture].

"Since development of iPS cells, it has become possible to convert cell types through altering epigenome by transducing plural kye transcription factors into cells. We found that transduction of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) into mouse dermal fibroblasts results in direct induction of chondrogeni cells. Directly induced chondrogenic cells showed highly methylated promoter sequences of fibroblastic markers, type I collagen genes. These induced cells, when transplanted into nude mice, produced hyaline cartilage without expressing type I collagen, suggesting that type I collagen genes were silenced in these induced chondrogenic cells in vivo ."

"the induced pluripotent stem cells (iPS cells), allowing creation of patient- and disease-specific stem cells. Collectively, the multipotency, high proliferation rates, and accessibility make the dental stem cell an attractive source of mesenchymal stem cells for tissue regeneration."

"PS cells resemble human ESC and can differentiate into advanced derivates of all three primary germ layers. Unlike ESC, iPS cell technology can derive patient-specific stem cells allowing derivation of tissue-matched differentiation donor cells for basic research, disease modeling, and regenerative medicine"

"mesenchymal stem cells are delivered into root canal spaces during regenerative endodontic procedures in immature teeth with open apices"

"in vitro de-differentiation of chondrocytes into fibrochondrocytes [occurs], which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells."

"Fibrocartilage is a type of scar tissue that expresses types I and II collagen; hyaline cartilage, in contrast, does not express type I collagen"

"The expression of a defined set of factors (Oct3/4, Sox2, c-Myc, and Klf4, as well as Nanog and human LIN28) can fully reprogram dermal fibroblasts into iPS cells"

"Primary chondrocytes expressed a small amount of Col1a1 and Col1a2, probably due to minor contamination of fibroblasts during the harvesting procedure or de-differentiation of chondrocytes"

"The cytosine guanine (CpG) dinucleotides in the promoters of the fibroblast-associated genes Col1a1 and Col1a2 were highly methylated in MK cell lines, but were unmethylated in parental MDFs. This suggests that expression of c-Myc, Klf4, and SOX9 induced silencing of the Col1a1 and Col1a2 genes in MDF culture. The Col1a1 promoter was moderately methylated in primary chondrocytes. Despite the low expression levels of Col1a2, its promoter was not methylated in primary chondrocytes prepared from neonatal rib cartilage. These results suggest that fibroblast marker gene promoters were excessively methylated in induced cells as compared with those in primary chondrocytes."

"the absence of type I collagen is prerequisite for proper function of hyaline cartilage"

Cell sources for cartilage repair; contribution of the mesenchymal perivascular niche.

"Tissue and cell sources for cartilage repair are revised, including: 1) cartilage and subchondral bone (auto and allografts; single or multiple/mosaicplasty grafts), 2) cultured chondrocytes (autologous/ACI, characterized/CCI, matrix assisted/MAC, or allogenic), 3) adult mesenchymal stem cells (MSCs), 4) progenitor cells from perichondrium and periosteum, 5) embryonic and prenatal stem cells, 6) induced pluripotent stem cells, and 7) genetically modified cells. We consider the biological mechanisms that explain usage and possible complications, advantages and limitations, emerging technologies and possible modulations on extracellular matrix properties and on migration, proliferation, de-differentiation, re-differentiation, morphology, function and integration of the cells. The study of MSC role involve: a) identification, b) location (perivascular niche hypothesis, pericytes as progenitor cells), c) lineage (myoadipofibrogenic system: transit amplifying cells, fibroblast/myofibroblasts, chondrocytes, osteoblasts, odontoblasts, vascular smooth muscle cells and adipocytes), and d) use in cartilage repair, comprising: 1) MSCs recruited from neighbouring tissues (bone marrow stimulation, MSCs based "in situ" cartilage repair, microfracture) and 2) MSCs cultured and expanded from bone marrow, adipose tissue, synovial membrane or granulation tissue."

"The criteria for identification of human MSCs include the following: a) adherence to plastic in standard culture conditions, b) expression of at least CD-73, CD-90, and CD-105, while CD-11b, CD-14, CD-19, CD-34, CD-45, and CD-79a are negative, and c) "in vitro" differentiation into chondroblasts, adipocytes, and osteoblasts"

"n the bone marrow microvasculature there is a continuous layer of subendothelial pericytes, which acquires a reticular morphology (reticular cells [type III collagen expressing cells]) in the venous side. Therefore, the marrow pericytes may be the same entity as the bone marrow stromal cells, since they share features such as: a) similar location of pericytes and stromal cells, b) expression of similar markers, such as SMA, PDGFR beta, EGFR, and CD146, and c) similar response to growth factors"

" the repair sequence includes stages of granulation tissue formation: hematoma (fibrin-deposition binding of platelets), macrophage recruitment, angiogenesis (neovascularization), recruitment and proliferation of multipotent mesenchymal stromal cells, re-absorption of the fibrin clot, and development of a vascularized scar-like tissue"

"spontaneous differentiation and remodelling mainly result in a fibrocartilaginous repair tissue, which may be subjected to excessive deformation with mechanical failure and degeneration after 20-48 weeks"

"the new collagen does not project into or intermingle with the native cartilage, thus hampering the integration and adherence of the newly generated cartilage {maybe because the cells have a different origin}. Indeed, the results after microfracture in the knee and their comparison with ACI demonstrate problems regarding the durability of the repair tissue in major defects and in defects located in areas other than the femoral condyles. Covers that trap the cells in the initial stages of granulation tissue formation (preventing escape of cells and anabolic cell mediators from the site of repair, since fibrin deposition contains the highest percentage of migrating mesenchymal stem cells) have been developed (e.g. collagen matrix) "

"scaffolds (e.g. poli (DL) lactide-coglycoide or alginate-gelatin biopolymer hydrogel), cell-free or seeded with autologous chondrocytes, with osteochondral regenerative potential, have been developed experimentally, with restoration of hyaline cartilage and bone"

"Bone marrow aspirate[refers to removal of a sample of bone marrow via a needle] contains very few MSCs, which can be isolated by means of Stro-1+ antibody recognition "<-Adipose tissues tend to have more MSCs than bone marrow.

"differentiation (expression levels of the chondrocyte specific genes Sox9, collagen type II, aggrecan, and cartilage olygomeric matrix protein) was more prominent in cells cultured in collagen type II hydrogel and that it increased in a time dependent manner. In this way, to induce and maintain chondrogenesis, transforming growth factors (TGF) b1 and b3, fibroblast growth factor, bone morphogenic proteins (BMPs)-2, -6, and -9, and insulin-like growth factors, may be used "

"we implanted perforated rigid tubes in the rat soft tissue, generating a peritubular granulation[fibrous connective] tissue, which progresses through the holes reaching tube light. The granulation tissue evolved into connective  and adipose tissues, except in the intratubular zone near the tube wall close to the holes (in the angle formed between the inner surfaces of the hole and the tube wall), where, during contraction, the granulation tissue presses onto the rigid material, the cells differentiating into chondrocytes"

"The perichondrium and periosteum share the same origin, regulatory mechanisms, and some morphological and functional characteristics. The perichondrium may differentiate into the periosteum. Both perichondrium and periosteum produce multiple positive and negative factors regulating the differentiation of the underlying skeletal elements (e.g. regulating gene expression in the underlying chondrocytes). Both structures have two distinct morphologic layers, an outer fibrous layer and the cambium or inner cellular layer. The inner cellular layer contains fibroblasts and chondroprogenitor / osteoprogenitor cells (Multipotent periosteum cells). Therefore, the periosteum can promote new cartilage and its chondrogenic potential decreases with age"

"When the perichondrium or the periosteum were activated, and the pericytes of the local postcapillary venules were labelled with an exogenous marker, the process of cartilage and bone formation from chondrogenic and osteoprogenitor cells already present in the perichondrium and the periosteum was augmented by proliferation and differentiation of the labelled pericytes, which contributed a supplementary population of newly formed chondrocytes and osteoblasts"

"perichondrium and periosteum not only provide chondrogenic and osteoprogenitor cells but act as inducers of proliferation and differentiation of cells with mesenchymal capacity."

"scaffolds that facilitate environment for chondrogenesis [include] natural (collagen, fibrin, alginate, hyaluronan, agarose, chitosan) or synthetic materials that provide a biodegradable matrix with biochemical properties, supporting neomatrix deposition by chondrocytes, b) several signalling pathways and transcription factors (e.g.: Wnt, transforming growth factor β/bone morphogenetic protein signalling, PDGF, IGF-1, EGF, HGF), which act in migration (PDGF, IGF-1, EGF, HGF, TGF β), proliferation (EGF, PDG,F TGF β), and differentiation (dexamethasone, TGF β). Some may be locally introduced, modulating cell differentiation into cartilage, c) procedures to prevent escape of cells and anabolic cell mediators from the site of repair, d) strategies for cartilage integration, e) inhibition of cartilage degeneration and inflammation (TNF-x and IL-1 application), and f) gene transfer for optimization of cell chondrogenic capacity."

"cells expressing bone formation cytokines, including over-expression of BMPs, have been developed"

Bone tissue engineering: current strategies and techniques--part II: Cell types.

"Sox9, Sox5, and Sox6 induce the differentiation of MSCs to chondrocytes."

"In the process of OB differentiation, Runx2, Sp7, and canonical Wnt signaling also inhibit the differentiation of mesenchymal cells into chondrocytes."

"osteogenic potential appears to be one of the last lineage commitment phenotypes to be lost [with passaging i.e. cell divisions]"

"fluid flow is also sufficient to induce BMSCs differentiation. Interstingly, investigators have demonstrated that even in the absence of dexamethasone, fluid flow enhances BMSC proliferation, ALP activity, osteopontin secretion, and calcium deposition compared with static controls."

"when cultured in the presence of estrogen, BMSCs were shown to have a marked decrease in apoptosis [due to elevated BCL2] and colonies become more robust and lived longer."

"BMSCs contain an EPC[endothelial progenitor cell] subpopulation [and] are capable of recruiting vasculogenic progenitors, endothelial cells, and pericyte-like cells when seeded onto a ceramic scaffold."

"PSC[Periosteal Stem Cells] proliferated faster than BMSCs and that BMSCs were more osteogenic than PSC."<-thus perhaps PSCs are more chondrogenic.

Generation of human induced pluripotent stem cells from osteoarthritis patient-derived synovial cells.

"Human synovial cells isolated from two 71-year-old women with advanced OA were characterized and reprogrammed into induced PSCs by ectopic expression of 4 transcription factors (Oct-4, SOX2, Klf4, and c-Myc). The pluripotency status of each induced PSC line was validated by comparison with human embryonic stem cells (ESCs).
We found that OA patient-derived human synovial cells had human mesenchymal stem cell (MSC)-like characteristics, as indicated by the expression of specific markers, including CD14-, CD19-, CD34-, CD45-, CD44+, CD51+, CD90+, CD105+, and CD147+. "

"19,771 genes (44.96%) showed ≥2-fold differences in expression level in human induced PSCs [versus] human MSCs."

"Human ESC–specific genes (TDGF3, CLDN6, DPPA4, LEFTY1, SOX2, ZFP42, CKMT1B, POU5F1, and Nanog) were significantly up-regulated by ≥180 times in human induced PSCs compared to MSCs, while lineage-specific genes were down-regulated in human induced PSCs"

"human MSCs from patients with OA showed rapid induction of the hypertrophic marker type X collagen (COL10A1) "

"expression of aggrecan mRNA in chondrogenically differentiated human induced PSCs was markedly higher than that in human ESCs."

Genes upregulated in iPSCs versus MSCs also upregulated in LSJL:


In vitro modeling of paraxial mesodermal progenitors derived from induced pluripotent stem cells.

"We established a protocol for the differentiation of mouse iPS cells into paraxial mesodermal lineages in serum-free culture. The protocol was dependent on Activin signaling in addition to BMP and Wnt signaling which were previously shown to be effective for mouse ES cell differentiation. Independently of the cell origin, the number of transgenes, or the type of vectors used to generate iPS cells, the use of serum-free monolayer culture stimulated with a combination of BMP4, Activin A, and LiCl enabled preferential promotion of mouse iPS cells to a PDGFR-α(+)/Flk-1(-) population, which represents a paraxial mesodermal lineage. The mouse iPS cell-derived paraxial mesodermal cells exhibited differentiation potential into osteogenic, chondrogenic, and myogenic cells both in vitro and in vivo and contributed to muscle regeneration. Moreover, purification of the PDGFR-α(+)/KDR(-) population after differentiation allowed enrichment of human iPS cell populations with paraxial mesodermal characteristics. The resultant PDGFR-α(+)/KDR(-) population derived from human iPS cells specifically exhibited osteogenic, chondrogenic, and myogenic differentiation potential in vitro, implying generation of paraxial mesodermal progenitors similar to mouse iPS cell-derived progenitors."

"Activin A prevented apoptosis [of iPS cells]"

"the addition of high dose of BMP4 enhanced cell proliferation [and] the absence of BMP4 resulted in large apoptosis"

"LiCl enhances Wnt signaling by translocation of β-catenin from cytoplasm to nucleus followed by inhibiting the activity of GSK3β"

"Ectopic cartilage was covered with a capsule and did not form part of the teratoma since it did not contain any other tissues. the ectopic cartilage was derived from engrafted cells that expressed DsRed."

"we transplanted both PDGFR-α+ and PDGFR-α− cells (derived from DsRed/iPS cells) into the tibial anterior (TA) muscle of immunodeficient mice. Four weeks after transplantation, tumor formation was only observed in TA muscle engrafted with PDGFR-α− cells (n = 3)"  Go to the study and 5B to see the ectopic cartilage.

"The PSP and DP populations gave rise to Alcian Blue-positive chondrocytes, while the DN and KSP populations had very low chondrogenic potentials"

"Expression profile of PDGFR-α and KDR in differentiated human iPS cells on day 6. DP, double-positive population; DN, double-negative population; PSP, PDGFR-α single-positive population; KSP, KDR single-positive population." So you want PDGFRA positive for chondrogenesis.

"the addition of LiCl, which activates Wnt signaling via inhibition of GSK3β, dramatically increased the proportion of PDGFR-α+ cells in serum-free culture."

Cartilage tissue engineering using differentiated and purified iPSCs

"iPSCs derived from adult mouse fibroblasts were chondrogenically differentiated and purified by type II collagen (Col2)-driven green fluorescent protein (GFP) expression. Col2 and aggrecan gene expression levels were significantly up-regulated in GFP+ cells compared with GFP- cells and decreased with monolayer expansion. An in vitro cartilage defect model was used to demonstrate integrative repair by GFP+ cells seeded in agarose, supporting their potential use in cartilage therapies. In chondrogenic pellet culture, cells synthesized cartilage-specific matrix as indicated by high levels of glycosaminoglycans and type II collagen and low levels of type I and type X collagen. The feasibility of cell expansion after initial differentiation was illustrated by homogenous matrix deposition in pellets from twice-passaged GFP+ cells.  Increased microscale elastic moduli [was] associated with collagen alignment at the periphery of pellets, mimicking zonal variation in native cartilage."

From John Friedline:
"Micromass cell cultures of iPSCs with BMP-4 and dexamethasone.  Then the Col2 promoter/enhancer was utilized to enhance production of green fluorescent protein (GFP), which served as a marker for chrondrogenic differentiation within the iPSC population. Those iPSCs which did not chondrogenically differentiate(despite BMP-4 and dexamethasone exposure) were GFP negative.  The GFP+ cells expressed type II cartilage, Col2 and aggrecan typical of chondrocytes, whereas GFP- cells expressed very little of these markers.  Subsequently both the GFP+ and GFP- iPSCs were induced to proliferate in monolayer using FBS and bFGF.  After monolayer expansion, both sets of cells were centrifuged to form pellets and were cultured for 21 days with TGF-beta3, which enhanced production of GAG (glycosaminoglycans) in the GFP+ cells. "

"the inner half of the cartilagenous matrix from GFP+ cells had a much higher degree of elastic modulus than that produced by GFP- cells."

The iPSCs had an interstitial deletion from 2D to 2F3 in chromosome 2 and the loss of the Y sex chromosome.  Which is interesting considering that the pro-chondrogenic gene Sox9 is a sex-linked gene. According to Table S1, Sox9 was much more homologously expressed between GFP+ and GFP- cells than Col2, Agc, Col10, Col1, and Nanog. Col2, ColX, and Agc ranged from 10 to 100 fold difference whereas Sox9 was only 2 fold different.  Perhaps Sox9 was not the driving transcription factor behind chondrogenesis, Klf4 is a likely suspect.
According to Generation of Porcine-Induced Pluripotent Stem Cells by Using OCT4 and KLF4 Porcine Factors., BMP4 is a stem-cell like marker and BMP4 may activate Myc.  This may explain why BMP4 was most effective at inducing chondrogenesis given the transgenic expression of the four key stemness genes(Oct4, Sox2, Klf4, Myc).
Other BMP's were related to the stemness cluster according to String.Embl but BMP4 was the closest.  The alternative protein they mention TGFB3 was not able to be connected to the stemness cluster even at numerous steps away.
The scientists showed that the elastic modulus was higher for the GFP+ than the GFP- cells. Since both cells were in the same culture and exposed to the same stimulii,  perhaps the elastic modulus is indicative of actin cytoskeleton differences.  According to Actin filaments play a primary role for structural integrity and viscoelastic response in cells., a decrease in actin cytoskeleton organization is associated with a decrease in elasticity and viscosity.  But this difference was only for the inner region of pellets and not the outer region.  Structural indentation stiffness was higher for GFP+ over GFP- cells in bulk measurements.

Small molecule mesengenic induction of human induced pluripotent stem cells to generate mesenchymal stem/stromal cells.

"We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2{Lefty2 is downregulated by LSJL}). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin{up} and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells."

"SB431542 inhibits activation of the activin receptor-like kinase (ALK) receptors 4, 5, and 7, key members of the TGF-β signaling pathway, and is thought to induce hESC differentiation by inhibiting SMAD2/3 phosphorylation"

"exogenous transcription factors used to induce iPSC generation (OCT4, SOX2, and cMyc) typically suppress TGF-β superfamily signaling and KLF4"<-This is is consistent as KLF4 has similarities to Sox9 which is a target of TGFB.

Genetically Matched Human iPS Cells Reveal that Propensity for Cartilage and Bone Differentiation Differs with Clones, not Cell Type of Origin.

"Genetically matched human iPSCs from different origins were generated using bone marrow stromal cells (BMSCs) and dermal fibroblasts (DFs) of the same donor, and global gene expression profile, DNA methylation status, and differentiation properties into the chondrogenic and osteogenic lineage of each clone were analyzed. Although genome-wide profiling of DNA methylation suggested tissue memory in iPSCs, genes expressed differentially in BMSCs and DFs were equally silenced in our bona fide iPSCs. After cell-autonomous and induced differentiation, each iPSC clone exhibited various differentiation properties, which did not correlate with cell-of-origin.
The reprogramming process may remove the difference between DFs and BMSCs at least for chondrogenic and osteogenic differentiation."

"[DF] cells were separately transfected with pMXs vectors harboring four human Yamanaka factors (OCT3/4, SOX2, KLF4, and c-MYC) with FuGENE 6 transfection reagent"

"Using probe sets of differentially methylated regions (DMR) between DFs and BMSCs (6,176 in 485,531 probes), the overall methylation patterns of iPSC clones were similar to each other and those of ESCs, and distinct from those of original DFs and BMSCs"

Genes upregulated in BMSC's versus dermal fibroblasts also upregulated in LSJL:


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