ABO gene editing for the conversion of blood type A to universal type O in Rhnull donor-derived human-induced pluripotent stem cells.

The limited availability of red cells with extremely rare blood group phenotypes is one of the global challenges in transfusion medicine that has prompted the search for alternative self-renewable pluripotent cell sources for the in vitro generation of red cells with rare blood group types. One such phenotype is the Rhnull , which lacks all the Rh antigens on the red cell membrane and represents one of the rarest blood types in the world with only a few active blood donors available worldwide. Rhnull red cells are critical for the transfusion of immunized patients carrying the same phenotype, besides its utility in the diagnosis of Rh alloimmunization when a high-prevalence Rh specificity is suspected in a patient or a pregnant woman. In both scenarios, the potential use of human-induced pluripotent stem cell (hiPSC)-derived Rhnull red cells is also dependent on ABO compatibility. Here, we present a CRISPR/Cas9-mediated ABO gene edition strategy for the conversion of blood type A to universal type O, which we have applied to an Rhnull donor-derived hiPSC line, originally carrying blood group A. This work provides a paradigmatic example of an approach potentially applicable to other hiPSC lines derived from rare blood donors not carrying blood type O.

Integration-free induced pluripotent stem cells derived from a patient with autosomal recessive Alport syndrome (ARAS).

A skin biopsy was obtained from a 25-year-old female patient with autosomal recessive Alport syndrome (ARAS) with the homozygous COL4A3 mutation c.345delG, p.(P166Lfs*37). Dermal fibroblasts were derived and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53shRNA. The generated induced Pluripotent Stem Cell (iPSC) clone AS FiPS1 Ep6F-2 was free of genomically integrated reprogramming genes, had the specific homozygous mutation, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. This iPSC line offers a useful resource to study Alport syndrome pathomechanisms and drug testing.

Generation of integration-free induced pluripotent stem cell lines derived from two patients with X-linked Alport syndrome (XLAS).

Skin biopsies were obtained from two male patients with X-linked Alport syndrome (XLAS) with hemizygous COL4A5 mutations in exon 41 or exon 46. Dermal fibroblasts were extracted and reprogrammed by nucleofection with episomal plasmids carrying OCT3/4, SOX2, KLF4 LIN28, L-MYC and p53 shRNA. The generated induced Pluripotent Stem Cell (iPSC) lines AS-FiPS2-Ep6F-28 and AS-FiPS3-Ep6F-9 were free of genomically integrated reprogramming genes, had the specific mutations, a stable karyotype, expressed pluripotency markers and generated embryoid bodies which were differentiated towards the three germ layers in vitro. These iPSC lines offer a useful resource to study Alport syndrome pathomechanisms and drug testing.

M-cadherin-mediated intercellular interactions activate satellite cell division.

Adult muscle stem cells and their committed myogenic precursors, commonly referred to as the satellite cell population, are involved in both muscle growth after birth and regeneration after damage. It has been previously proposed that, under these circumstances, satellite cells first become activated, divide and differentiate, and only later fuse to the existing myofiber through M-cadherin-mediated intercellular interactions. Our data show that satellite cells fuse with the myofiber concomitantly to cell division, and only when the nuclei of the daughter cells are inside the myofiber, do they complete the process of differentiation. Here we demonstrate that M-cadherin plays an important role in cell-to-cell recognition and fusion, and is crucial for cell division activation. Treatment of satellite cells with M-cadherin in vitro stimulates cell division, whereas addition of anti-M-cadherin antibodies reduces the cell division rate. Our results suggest an alternative model for the contribution of satellite cells to muscle development, which might be useful in understanding muscle regeneration, as well as muscle-related dystrophies.

Generation of induced pluripotent stem cells from human renal proximal tubular cells with only two transcription factors, OCT4 and SOX2.

The tubular epithelium of the kidney is susceptible to injury from a number of different causes, including inflammatory and immune disorders, oxidative stress, and nephrotoxins, among others. Primary renal epithelial cells remain one of the few tools for studying the biochemical and physiological characteristics of the renal tubular system. Nevertheless, differentiated primary cells are not suitable for recapitulation of disease properties that might arise during embryonic kidney formation and further maturation. Thus, cellular systems resembling kidney characteristics are in urgent need to model disease as well as to establish reliable drug-testing platforms. Induced pluripotent stem cells (iPSCs) bear the capacity to differentiate into every cell lineage comprising the adult organism. Thus, iPSCs bring the possibility for recapitulating embryonic development by directed differentiation into specific lineages. iPSC differentiation ultimately allows for both disease modeling in vitro and the production of cellular products with potential for regenerative medicine. Here, we describe the rapid, reproducible, and highly efficient generation of iPSCs derived from endogenous kidney tubular renal epithelial cells with only two transcriptional factors, OCT4 and SOX2. Kidney-derived iPSCs may provide a reliable cellular platform for the development of kidney differentiation protocols allowing drug discovery studies and the study of kidney pathology.

Role of versican V0/V1 and CD44 in the regulation of human melanoma cell behavior.

Versican is a hyaluronan-binding, large extracellular matrix chondroitin sulfate proteoglycan whose expression is increased in malignant melanoma. Binding to hyaluronan allows versican to indirectly interact with the hyaluronan cell surface receptor CD44. The aim of this work was to study the effect of silencing the large versican isoforms (V0 and V1) and CD44 in the SK-mel-131 human melanoma cell line. Versican V0/V1 or CD44 silencing caused a decrease in cell proliferation and migration, both in wound healing assays and in Transwell chambers. Versican V0/V1 silencing also caused an increased adhesion to type I collagen, laminin and fibronectin. These results support the proposed role of versican as a proliferative, anti-adhesive and pro-migratory molecule. On the other hand, CD44 silencing caused a decrease in cell adhesion to vitronectin, fibronectin and hyaluronan. CD44 silencing inhibited the binding of a FITC-hyaluronan complex to the cell surface and its internalization into the cytoplasm. Our results indicate that both versican and CD44 play an important role regulating the behavior of malignant melanoma cells.

V3 versican isoform alters the behavior of human melanoma cells by interfering with CD44/ErbB-dependent signaling.

Versican is a hyaluronan-binding, extracellular chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The short V3 isoform contains the G1 and G3 terminal domains of versican that may potentially interact directly or indirectly with the hyaluronan receptor CD44 and the EGFR, respectively. We have previously described that overexpression of V3 in MeWo human melanoma cells markedly reduces tumor cell growth in vitro and in vivo. In this study we have investigated the signaling mechanism of V3 by silencing the expression of CD44 in control and V3-expressing melanoma cells. Suppression of CD44 had the same effects on cell proliferation and cell migration than those provoked by V3 expression, suggesting that V3 acts through a CD44-mediated mechanism. Furthermore, CD44-dependent hyaluronan internalization was blocked by V3 expression and CD44 silencing, leading to an accumulation of this glycosaminoglycan in the pericellular matrix and to changes in cell migration on hyaluronan. Furthermore, ERK1/2 and p38 activation after EGF treatment were decreased in V3-expressing cells suggesting that V3 may also interact with the EGFR through its G3 domain. The existence of a EGFR/ErbB2 receptor complex able to interact with CD44 was identified in MeWo melanoma cells. V3 overexpression resulted in a reduced interaction between EGFR/ErbB2 and CD44 in response to EGF treatment. Our results indicate that the V3 isoform of versican interferes with CD44 and the CD44-EGFR/ErbB2 interaction, altering the signaling pathways, such as ERK1/2 and p38 MAPK, that regulate cell proliferation and migration.

Differential expression of endoglin in human melanoma cells expressing the V3 isoform of versican by microarray analysis.

Versican is a large chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The large isoforms V0 and V1 promote melanoma cell proliferation. We previously described that overexpression of the short V3 isoform in MeWo human melanoma cells markedly reduced tumor cell growth in vitro and in vivo, but favored the appearance of secondary tumors. This study aimed to elucidate the mechanisms of V3 by identifying differentially expressed genes between parental and V3-expressing MeWo melanoma cells using microarray analysis. V3 expression significantly reduced the expression of endoglin, a transforming growth factor-ß superfamily co-receptor. Other differentially expressed genes were VEGF and PPP1R14B. Changes in endoglin levels were validated by qRT-PCR and Western blotting.

Structure and regulation of the versican promoter: the versican promoter is regulated by AP-1 and TCF transcription factors in invasive human melanoma cells.

Versican is a large chondroitin sulfate proteoglycan of the extracellular matrix that is involved in a variety of cellular processes. We showed previously that versican, which is overexpressed in cutaneous melanomas as well as in premalignant lesions, contributes to melanoma progression, favoring the detachment of cells and the metastatic dissemination. Here, we investigated the transcriptional regulation of the versican promoter in melanoma cell lines with different levels of biological aggressiveness and stages of differentiation. We show that versican promoter up-regulation accounts for the differential expression levels of mRNA and protein detected in the invasive SK-mel-131 human melanoma cells. The activity of the versican promoter increased 5-fold in these cells in comparison with that measured in non-invasive MeWo melanoma cells. Several transcriptional regulatory elements were identified in the proximal promoter, including AP-1, Sp1, AP-2, and two TCF-4 sites. We show that promoter activation is mediated by the ERK/MAPK and JNK signaling pathways acting on the AP-1 site, suggesting that BRAF mutation present in SK-mel-131 cells impinge upon the up-regulation of the versican gene through signaling elicited by the ERK/MAPK pathway. This is the first time the AP-1 transcription factor family has been shown to be related to the regulation of versican expression. Furthermore, deletion of the TCF-4 binding sites caused a 60% decrease in the promoter activity in SK-mel-131 cells. These results showing that AP-1 and TCF-4 binding sites are the main regulatory regions directing versican production provide new insights into versican promoter regulation during melanoma progression.

Altered expression of versican and hyaluronan in melanocytic tumors of dogs.

OBJECTIVE: To analyze the expression of versican and hyaluronan in melanocytomas and malignant melanomas of dogs, to correlate their expression with expression of the hyaluronan receptor CD44, and to identify enzymes responsible for the synthesis and degradation of hyaluronan in canine dermal fibroblasts and canine melanoma cell lines. SAMPLE POPULATION: 35 biopsy specimens from melanocytic tumors of dogs, canine primary dermal fibroblasts, and 3 canine melanoma cell lines. PROCEDURES: Versican, hyaluronan, and CD44 were detected in tumor samples by use of histochemical or immunohistochemical methods. Expression of hyaluronan-metabolizing enzymes was analyzed with a reverse transcriptase-PCR assay. RESULTS: Versican was found only in some hair follicles and around some blood vessels in normal canine skin, whereas hyaluronan was primarily found within the dermis. Hyaluronan was found in connective tissue of the oral mucosa. Versican and, to a lesser extent, hyaluronan were significantly overexpressed in malignant melanomas, compared with expression in melanocytomas. No significant difference was found between malignant tumors from oral or cutaneous origin. The expression of both molecules was correlated, but hyaluronan had a more extensive distribution than versican. Versican and hyaluronan were mainly associated with tumor stroma. Canine fibroblasts and melanoma cell lines expressed hyaluronan synthase 2 and 3 (but not 1) and hyaluronidase 1 and 2. CONCLUSIONS AND CLINICAL RELEVANCE: Versican may be useful as a diagnostic marker for melanocytic tumors in dogs. Knowledge of the enzymes involved in hyaluronan metabolism could reveal new potential therapeutic targets.

V3 versican isoform expression has a dual role in human melanoma tumor growth and metastasis.

Versican is a large chondroitin sulfate proteoglycan produced by several tumor cell types, including malignant melanoma, which exists as four different splice variants. The presence of versican in the extracellular matrix plays a role in tumor cell growth, adhesion and migration, which could be altered by altering the ratio between versican isoforms. We have previously shown that overexpression of the V3 isoform of versican in human melanoma cell lines markedly reduces cell growth in vitro and in vivo, since V3-overexpressing (LV3SN) cultured cells as well as primary tumors arising from these cells grow slower than their vector-only counterparts (LXSN). In the present work, we have extended these observations to demonstrate that the delayed cell growth is due to multiple events since differences in proliferative index as well as in apoptosis are observed in LV3SN cells and tumors compared to LXSN. For example, LV3SN melanoma cells exhibit delayed activation of MAPK in response to EGF, we have also characterized further the primary tumors originated in nude mice from V3-transduced melanoma cells to determine if other events affect the V3 tumor phenotype. For example, hyaluronan content of LV3SN tumors was higher than in LXSN tumors, whereas other related matrix components and vascularization were unaffected. Furthermore, lung metastasis in nude mice occurred only in animals carrying LV3SN tumors, indicating a dual role for this molecule, both as an inhibitor of tumor growth and a metastasis inductor.