Publicaciones

Publicado en: Nature Communications

Abstract. Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche.

Referencia:

Chao Wang, Ruben Rabadan Ros, Paloma Martinez-Redondo, Zaijun Ma, Lei Shi, Yuan Xue, Isabel Guillen-Guillen, Ling Huang, Tomoaki Hishida, Hsin-Kai Liao, Estrella Nuñez Delicado, Concepcion Rodriguez Esteban, Pedro Guillen-Garcia, Pradeep Reddy & Juan Carlos Izpisua Belmonte

Link: https://doi.org/10.1038/s41467-021-23353-z

Publicado en: Protein Cell

Martinez-Redondo, P., Guillen-Guillen, I., Davidsohn, N. et al. αKLOTHO and sTGFβR2 treatment counteract the osteoarthritic phenotype developed in a rat model. Protein Cell

Homeostasis and repair are critical biological processes that allow for tissue and organ preservation and function in multi-cellular organisms. Their regulation and extension vary drastically across the animal kingdom, and mammals show limited tissue-specific regenerative capacity that declines with age. During aging, articular cartilage is one of the tissues that undergo substantial changes in the matrix structure, molecular composition, metabolic activity, and mechanical properties (Loeser et al. 2016). As a result, articular cartilage experiences impaired homeostasis and limited capacity to undergo repair, contributing to osteoarthritis (OA) development (Loeser et al. 2016). OA is the most prevalent musculoskeletal disorder among the elderly and is the leading cause of disability in the US due to pain associated with the disease (Zhang et al. 2016). Although symptomatic pain relief is possible (Zhang et al. 2016), treatments to cure the pathology are currently unavailable. Interestingly, contrary to the loss of homeostasis and repair capacity with age, during embryogenesis as well as a short period after birth, mammals seem to have a higher regeneration capacity (Vivien et al. 2016). These and other facts beg the question of whether therapeutic targets can be developed towards the enhancement of the low regenerative capacity observed during adulthood and worsen upon aging.

Referencia:

Paloma Martinez-Redondo, Isabel Guillen-Guillen, Chao Wang, Javier Prieto, Masakazu Kurita, Fumiyuki Hatanaka, Cuiqing Zhong, Reyna Hernandez-Benitez, Tomoaki Hishida, Takashi Lezaki, Akihisa Sakamoto, Amy N. Nemeth, Yuriko Hishida, Concepcion Rodriguez Esteban, Kensaku Shojima, Pradeep Reddy & Juan Carlos Izpisua Belmonte

Paloma Martinez-Redondo and Isabel Guillen-Guillen have contributed equally to this work.

Link: https://link.springer.com/article/10.1007%2Fs13238-019-00685-7

Publicado en: Revista Española de Artroscopia y Cirugía Articular (REACA)

La medicina regenerativa es una nueva disciplina de la medicina, con un futuro muy prometedor para el tratamiento de muchas dolencias que aquejan al ser humano. Para muchas enfermedades todavía está en fase de experimentación. Uno de los campos en los que más ha avanzado es en el tratamiento del cartílago, desde la utilización de cultivos de condrocitos autólogos en medio líquido, al empleo de biomateriales como la membrana de colágeno I/III y, finalmente, al aumento de dosis celular en la técnica ICC. Por ello, en las lesiones de cartílago de grado III/IV de Outerbridge de rodilla, tobillo y ahora ya en cadera, podemos usar la técnica de implante de condrocitos en membrana (ICC), consiguiendo la regeneración del tejido cartilaginoso. Se trata de medicina regenerativa, pues se forma un tejido cartilaginoso de naturaleza hialina con la misma estructura y función que el cartílago articular sano. Además, los pacientes presentan una recuperación funcional excelente, consiguiendo la vuelta al deporte en muchas ocasiones.

Referencia:

Isabel Guillén Vicente, Marta Guillén Vicente, Lucía Aboli Martínez, Juan Manuel López Alcorocho, María Dolores Pérez Pérez, Ramón Navarro Mont, Ana Belén Abenoja Lobo, Elena Rodríguez Íñigo, Pedro Guillén García

Link: https://fondoscience.com/sites/default/files/articles/pdf/reaca.28171.fs1909039-instant-cemtro-cell-icc-implante-condrocitos.pdf

Publicado en: Nature Communications

Abstract. DiGeorge syndrome critical region 8 (DGCR8) is a critical component of the canonical microprocessor complex for microRNA biogenesis. However, the non-canonical functions of DGCR8 have not been studied. Here, we demonstrate that DGCR8 plays an important role in maintaining heterochromatin organization and attenuating aging. An N-terminal-truncated version of DGCR8 (DR8^dex2) accelerated senescence in human mesenchymal stem cells (hMSCs) independent of its microRNA-processing activity. Further studies revealed that DGCR8 maintained heterochromatin organization by interacting with the nuclear envelope protein Lamin B1, and heterochromatin-associated proteins, KAP1 and HP1γ. Overexpression of any of these proteins, including DGCR8, reversed premature senescent phenotypes in DR8^dex2 hMSCs. Finally, DGCR8 was downregulated in pathologically and naturally aged hMSCs, whereas DGCR8 overexpression alleviated hMSC aging and mouse osteoarthritis. Taken together, these analyses uncovered a novel, microRNA processing-independent role in maintaining heterochromatin organization and attenuating senescence by DGCR8, thus representing a new therapeutic target for alleviating human aging-related disorders.

Referencia:

Liping Deng, Ruotong Ren, Zunpeng Liu, Moshi Song, Jingyi Li, Zeming Wu, Xiaoqing Ren, Lina Fu, Wei Li, Weiqi Zhang, Pedro Guillen, Juan Carlos Izpisua Belmonte, Piu Chan, Jing Qu & Guang-Hui Liu

Link: https://www.nature.com/articles/s41467-019-10831-8

Publicado en: Cartilage

Purpose. Two-year follow-up to assess efficacy and safety of high-density autologous chondrocyte implantation (HD-ACI) in patients with cartilage lesions in the ankle. Design. Twenty-four consecutive patients with International Cartilage repair Society (ICRS) grade 3-4 cartilage lesions of the ankle were included. Five million chondrocytes per cm2 of lesion were implanted using a type I/III collagen membrane as a carrier and treatment effectiveness was assessed by evaluating pain with the visual analogue scale (VAS) and American Orthopaedic Foot & Ankle Society (AOFAS) ankle-hindfoot score at baseline, 12-month, and 24-month follow-up, together with dorsal and plantar flexion. Magnetic resonance observation for cartilage repair tissue (MOCART) score was used to evaluate cartilage healing. Histological study was possible in 5 cases. Results. Patients’ median age was 31 years (range 18-55 years). Median VAS score was 8 (range 5-10) at baseline, 1.5 (range 0-8) at 12-month follow-up, and 2 (rang e0-5) at 24-month follow-up (P < 0.001). Median AOFAS score was 39.5 (range 29-48) at baseline, 90 (range 38-100) at 12-month follow-up, and 90 (range 40-100) at 24-month follow-up (P < 0.001). Complete dorsal flexion significantly increased at 12 months (16/24, 66.7%) and 24 months (17/24, 70.8%) with regard to baseline (13/24, 54.2%) (P = 0.002). MOCART at 12- and 24-month follow-ups were 73.71 ± 15.99 and 72.33 ± 16.21. Histological study confirmed that neosynthetized tissue was cartilage with hyaline extracellular matrix and numerous viable chondrocytes. Conclusion. HD-ACI is a safe and effective technique to treat osteochondral lesions in the talus, providing good clinical and histological results at short- and mid-term follow-ups.

Referencia:

Juan Manuel López-Alcorocho, Isabel Guillén-Vicente, Elena Rodríguez-Iñigo, Ramón Navarro, Rosa Caballero-Santos, Marta Guillén-Vicente, Mercedes Casqueiro, Tomás F. Fernández-Jaén, Fernando Sanz, Santiago Arauz, Steve Abelow, and Pedro Guillén-García

Link: https://pubmed.ncbi.nlm.nih.gov/30880428/

Publicado en: CARTILAGE

Abstract. In the process of cell division, the extremes of the eukaryotic chromosomes are progressively shortening, and this phenomenon is related to cell degeneration and senescence. The treatment of cartilage lesions with autologous chondrocytes implies that cells proliferate in an artificial environment. We have studied the viability of cultured chondrocytes after measurement of their telomere length before implantation. Methods. Articular cartilage biopsies (B1, B2, and B3) were obtained from 3 patients (2 males and 1 female) with knee cartilage defects, who were going to be treated with chondrocyte implantation. Chondrocytes were cultured in DMEM with autologous serum. After the third passage, an aliquot of 1 million cells was removed to estimate the telomere length and the remaining cells were implanted. Telomere length was measured by quantitative fluorescent in situ hybridization (Q-FISH). Patients’ clinical outcome was determined preoperatively, and 12 and 24 months postimplantation with the International Knee Documentation Committee (IKDC) questionnaire. Results. After chondrocyte implantation, IKDC score doubled at 12 and 24 months with regard to the basal value. After 3 passages, chondrocytes were cultured for a mean of 45.67 days, the mean duplication time being 4.53 days and the mean number of cell divisions being 10.04 during the culture period. The 20th percentile of telomere lengths were 6.84, 6.96, and 7.06 kbp and the median telomere lengths 10.30, 10.47, and 10.73 kbp, respectively. No significant correlation was found between IKDC score and telomere length. Conclusion. Culturing autologous chondrocytes for implantation is not related to cell senescence in terms of telomere length.

Referencia:

Juan Manuel López-Alcorocho, Isabel Guillén-Vicente, Elena Rodríguez-Iñigo, Marta Guillén-Vicente, Tomás Fernando Fernández-Jaén, Rosa Caballero, Mercedes Casqueiro, Pilar Najarro, Steve Abelow, and Pedro Guillén-García

Link: https://pubmed.ncbi.nlm.nih.gov/29322876/

Publicado en: Cell

Summary

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases.

Highlights

• A CRISPR/Cas9 system transcriptionally activates endogenous target genes in vivo
• Recruiting the transcriptional machinery induces transepigenetic remodeling
• Inducing target gene expression leads to physiological phenotypes in postnatal mammals
• The system ameliorates symptoms associated with several mouse models of human diseases

Referencia:

• Hsin-Kai Liao, Fumiyuki Hatanaka,
• Toshikazu Araoka, …, Li-Fan Lu
• Concepcion Rodriguez Esteban
• Juan Carlos Izpisua Belmonte

Link: https://pubmed.ncbi.nlm.nih.gov/29224783/

Publicado en: Cartilage

Objective. The aim of this work was to study the short- and mid-term effectiveness and safety of high-density autologous chondrocyte implantation (HD-ACI) in the first 50 patients with knee cartilage damage treated in our unit. Design. Fifty consecutive patients with cartilage lesions (Outerbridge grade III-IV) in the knee treated with HD-ACI were included in this study. Chondrocytes were isolated from a nonbearing cartilage area biopsy and were cultured until 40 to 50 million cells were obtained. Five million chondrocytes per cm2 of a porcine collagen type I/III membrane were implanted covering the defect. Procedure effectiveness was assessed by evaluating pain, swelling, and range of mobility (flexion and extension) at 6-, 12-, and 24-month follow-up. The International Knee Documentation Committee (IKDC) subjective evaluation form was used to evaluate symptoms and functions of the knee. Results. The percentage of patients with pain and swelling decreased progressively in the following visits, with differences being statistically significant (P < 0.001 and P = 0.040, respectively). IKDC scores improved progressively throughout the 24-month follow-up (P < 0.001). Thus, the mean IKDC score improvement was 26.3 points (95% confidence interval [CI] = 18.2-34.4 points) at 12 months and 31.0 points (95% CI = 22.9-39 points) at 24 months. No significant differences were found when performing extension (P = 0.112). Flexion significantly improved by 25.1° at 24-month follow-up (P = 0.013). Conclusions. HD-ACI is a safe and effective technique for the treatment of cartilage defects, improving clinical and subjective perception of knee functionality. These preliminary results encourage future studies comparing this technique with traditional ACI.

Referencia:

J.M. López-Alcorocho, L. Aboli, I. Guillén-Vicente, E. Rodriguez Iñigo, M. Guillén-Vicente, T. F. Fdez.-Jaén, S. Arauz, S. Abelow and P. Guillén-García. “Cartilage Defect Treatment Using High-Density Autologous Chondrocyte Implantation: Two-Year Follow-up” Cartilage 1-7 DOI:10.1177/1947603517693045

Link: http://journals.sagepub.com/doi/abs/10.1177/1947603517693045

Publicado en: Cell

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Referencia:

J.Wu,A.Platero-Luengo, M.Sakurai, A. Sugawara, M. A. Gil, T. Yamauchi, K.Suzuki, Y.S. BGoglioti, C. Cuello,M. Morales, D. Okumura, J. Luo, M. Vilariño, I.Parrillo, D. A.Soto, C.A. Martinez, T. Hishida, S. Sanchez-Bautista, M. llanos Martinez, H. Wang, A. Nohalez, E. Aizawa, P. Martinez-Redondo, A. Ocampo, P. Reddy, J. Roca, E. A.Maga, C. Rodriguez Esteban,  W. Travis, E. Nuñez Delicado, J. Lajara, I.Guillén, P. Guillén, J. Campistol, E. A. Martinez, P. J. Ross an J.C. Izpisúa “Interspecies chimerism with mammalian pluripotent stem cells” Cell 168, 1-14 January 26, 2017.

Link: http://www.cell.com/cell/abstract/S0092-8674(16)31752-4?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867416317524%3Fshowall%3Dtrue

Publicado en: Nature

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient1, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders2. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)3, 4 technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Autores:

Suzuki, K., Tsunekawa, Y., Hernandez-Benitez, R., Wu, J., Zhu, J., Kim, E., Hatanaka, F., Yamamoto, M., Araoka, T., Li, Z., Kurita, M., Hishida, T., Li, M., Aizawa, E., Chen, S., Goebl, A., Soligalla, R.D., Qu, J., Jiang, T., Skowronska-Krawczyk, D., Rodriguez Esteban, C., Lajara, J., Nuñez, E., Guillen, P., Campistol, J.M., Matsuzaki, F., Liu, G.-H., Magistretti, P., Zhang, K., Callaway, E., Zhang, K and Izpisua Belmonte, J.C. “In vivo genome editing via CRISPR-Cas9 mediated homology-independent targeted integration” Nature 540, 144-149 (01 December 2016).

Link: http://www.nature.com/nature/journal/v540/n7631/full/nature20565.html