Enhanced Isolation of Fibroblasts from Human Skin Explants

Huschtscha, Lily I.; Napier, Christine E.; Noble, Jane R.; Bower, Kylie; Au, Amy Y M; Campbell, Heather; Braithwaite, Antony W.; Reddel, Roger R.

doi:10.2144/0000113939

Cited by 30 publications

(31 citation statements)

References 12 publications

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“…The clinical application of autologous MSC transplantation to bone regeneration faces some obstacles, however, especially in elderly patients, including limited numbers of stem cells, age-related decrease in potential for differentiation into osteoblasts, and invasion during the cell isolation procedure (41,42). Such obstacles may be avoided by applying the direct reprogramming technology, in which a large number of functional osteoblasts can be obtained from fibroblasts isolated from patients or allogenic donors by a less-invasive procedure (43,44). Fibroblasts have a high proliferative capacity, which does not decline with increasing donor age (43,45).…”

Section: Discussionmentioning

confidence: 99%

“…Such obstacles may be avoided by applying the direct reprogramming technology, in which a large number of functional osteoblasts can be obtained from fibroblasts isolated from patients or allogenic donors by a less-invasive procedure (43,44). Fibroblasts have a high proliferative capacity, which does not decline with increasing donor age (43,45). Thus, direct reprogramming may provide another option for such patients in whom MSC transplantation might not be an appropriate therapy.…”

Section: Discussionmentioning

confidence: 99%

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Direct conversion of human fibroblasts into functional osteoblasts by defined factors

Yamamoto

Kishida²,

Satô

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

108

103

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Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.O steoblasts play a central role in bone formation and remodeling by producing type I collagen, osteopontin, osteocalcin, and bone sialoprotein (BSP), and calcifying these bone matrixes (1). They are also involved in hematopoiesis, phosphate metabolism, and glucose metabolism (2). Osteoblasts are derived from mesenchymal progenitor cells that are common precursors shared by chondrocytes, adipocytes, and myoblasts (3). The differentiation of osteoblasts is regulated by various transcription factors, including Runt-related transcription factor 2 [Runx2, also known as core-binding factor subunit α-1 (Cbfα-1)] (4, 5), Osterix (6, 7), Distal-less homeobox 5 (Dlx5) (8), and activation transcription factor 4 (ATF-4) (8). A functional decline in osteoblasts relative to osteoclasts results in imbalance between bone formation and resorption and may cause osteolytic pathological conditions, such as osteoporosis (9), alveolar bone resorption associated with periodontitis (10), and bone lysing associated with bone tumors, including multiple myeloma (11).It has been demonstrated that forced expression of combinations of some transcription factors, such as Octamer-binding transcription factor 3/4 (Oct4), Sox2, Klf-4, and c-Myc (reprogramming factors), induces immortality and pluripotency in mammalian somatic cells (12, 13). The generation of induced pluripotent stem (iPS) cells clearly indicates that genome-wide epigenetic programming can be drastically changed in somatic cells by a small number of transcription factors that may have key regulatory roles in cell fate decisions (14,15).Recent studies have reported that direct conversion, or direct reprogramming, of somatic cells into another dif...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Direct conversion of human fibroblasts into functional osteoblasts by defined factors

Yamamoto

Kishida²,

Satô

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

108

103

View full text Add to dashboard Cite

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“…19 Skin was obtained during clubfoot surgery from patient 1 (HOXC deletion) and two reference samples without HOXC deletions.…”

Section: Human Skin Fibroblastsmentioning

confidence: 99%

Deletions of 5′HOXCgenes are associated with lower extremity malformations, including clubfoot and vertical talus

Alvarado

McCall

Hecht³

et al. 2016

J Med Genet

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Background Deletions of the HoxC gene cluster result in variable phenotypes in mice, but have been rarely described in humans. Here, we report chromosome 12q13.13 microdeletions ranging from 13-175 kb and involving the 5’ HOXC genes in four families segregating congenital lower limb malformations, including clubfoot, vertical talus, and hip dysplasia. Methods Probands (N=253) with clubfoot or vertical talus were screened for point mutations and copy number variants (CNVs) using Multiplexed Direct Genomic Selection (MDiGS), a pooled BAC targeted capture approach. SNP genotyping included 1178 clubfoot or vertical talus probands and 1775 controls. Results The microdeletions share a minimal noncoding region overlap upstream of HOXC13, with variable phenotypes depending upon HOXC13, HOXC12 or the HOTAIR lncRNA inclusion. SNP analysis revealed HOXC11 p.Ser191Phe segregating with clubfoot in a small family and enrichment of HOXC12 p.Asn176Lys in patients with clubfoot or vertical talus (rs189468720, p=0.0057, OR=3.8). Defects in limb morphogenesis include shortened and overlapping toes, as well as peroneus muscle hypoplasia. Finally, HOXC and HOXD gene expression is reduced in fibroblasts from a patient with a 5’ HOXC deletion, consistent with prior studies demonstrating that dosage of lncRNAs alters expression of HOXD genes in trans. Conclusions Because HOXD10 has been implicated in the etiology of congenital vertical talus, variation in its expression may contribute to the lower limb phenotypes occurring with 5’ HOXC microdeletions. Identification of 5’ HOXC microdeletions highlights the importance of transcriptional regulators in the etiology of severe lower limb malformations and will improve their diagnosis and management.

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“…32 Primary fibroblasts obtained from Vcan flox/ flox mice by explant culture 33 were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. QRsP11 cells were transfected with pCMV-tdTomato plasmid (Clontech Laboratories, Mountain View, CA), and treated with 300 lg/ml G418 treatment for 7 days, followed by cloning twice to obtain the stable clones designated as Q11-12.…”

Section: Cell Culturementioning

confidence: 99%

Host stromal versican is essential for cancer‐associated fibroblast function to inhibit cancer growth

Fanhchaksai

Okada

Nagai

et al. 2015

Intl Journal of Cancer

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The stroma provides a microenvironment that regulates tumor cell behavior. The extracellular matrix (ECM) has long been recognized to be important in tumor cell behavior, and previous studies have revealed the impact of individual matrix molecules on tumor progression. Although several reports have highlighted some central roles of tumor cell-expressed versican, the role of host stromal versican is not yet understood. In this study, we demonstrate that versican is an important molecule in the functional ECM structure and maintaining cancer-associated fibroblasts, using versican-negative QRsP11 fibrosarcoma cells. By their subcutaneous injection with cre-expressing adenoviruses to versican-floxed mice, we demonstrate that loss of host stromal versican facilitates tumor cell proliferation, and following angiogenesis, decreases cancer-associated fibroblasts, diminishes collagen fibers and alters hyaluronan distribution, concomitant with upregulation of hyaluronan, TGFb and VEGFmediated signaling. When the versican V3 variant consisting of G1 and G3 domains was expressed in tumor cells, it was integrated into the ECM, regained collagen fibers and cancer-associated fibroblasts and resulted in successful recovery of tumor growth inhibition, indicating that whatever cells produce, the G1 and G3 domains are adequate for versican function. Collectively, our results indicate a dynamic function of versican in the ECM that regulates tumor cell behavior. A greater understanding of the regulation of versican expression may contribute to the development of cancer therapies.Cancer cell behavior is largely affected by its microenvironment (such as the stroma), which comprises the extracellular matrix (ECM), fibroblasts and blood vessels.

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Enhanced Isolation of Fibroblasts from Human Skin Explants

Cited by 30 publications

References 12 publications

Direct conversion of human fibroblasts into functional osteoblasts by defined factors

Direct conversion of human fibroblasts into functional osteoblasts by defined factors

Deletions of 5′HOXCgenes are associated with lower extremity malformations, including clubfoot and vertical talus

Host stromal versican is essential for cancer‐associated fibroblast function to inhibit cancer growth

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