Diferenciación dirigida es una bioingeniería metodología en la interfase de la biología de células madre , la biología del desarrollo y la ingeniería de tejidos . [1] Básicamente, se trata de aprovechar el potencial de las células madre al restringir su diferenciación in vitro hacia un tipo de célula o tejido específico de interés. [2] Las células madre son, por definición , pluripotentes , capaces de diferenciarse en varios tipos de células como neuronas , [3] cardiomiocitos , hepatocitos , etc. EficienteLa diferenciación dirigida requiere una comprensión detallada del linaje y la decisión del destino celular , a menudo proporcionada por la biología del desarrollo. [2] [4]
Marco conceptual
Durante la diferenciación, las células pluripotentes toman una serie de decisiones de desarrollo para generar primero las tres capas germinales ( ectodermo , mesodermo y endodermo ) del embrión y los progenitores intermedios, [5] seguidas de decisiones posteriores o puntos de control, dando lugar a todas las capas maduras del cuerpo. tejidos. [4] El proceso de diferenciación se puede modelar como una secuencia de decisiones binarias basadas en modelos estocásticos o probabilísticos . La biología del desarrollo y la embriología proporcionan el conocimiento básico de la diferenciación de los tipos de células a través del análisis de mutaciones , rastreo de linajes, micromanipulación de embriones y estudios de expresión génica . La diferenciación celular y la organogénesis tisular implican un conjunto limitado de vías de señalización del desarrollo . [4] Por tanto, es posible dirigir el destino de la célula controlando las decisiones de la célula a través de la señalización extracelular, imitando las señales de desarrollo.
Material de origen
La diferenciación dirigida se aplica principalmente a células madre pluripotentes (PSC) de origen mamífero, en particular células de ratón y humanas para aplicaciones de investigación biomédica . [5] Desde el descubrimiento de las células madre embrionarias (ES) (1981) y las células madre pluripotentes inducidas (iPS) (2006), el material de origen es potencialmente ilimitado. [1] [4] [6] Históricamente, también se han utilizado células de carcinoma embrionario (CE). [7] Se han utilizado fibroblastos u otros tipos de células diferenciadas para estrategias de reprogramación directa . [1]
Métodos
La diferenciación celular implica una transición de un modo proliferativo a un modo de diferenciación. La diferenciación dirigida consiste en imitar las decisiones de desarrollo (desarrollo del embrión) in vitro utilizando las células madre como material de origen. [1] Para este propósito, las células madre pluripotentes (PSC) se cultivan en condiciones controladas que involucran sustratos específicos o matrices extracelulares que promueven la adhesión y diferenciación celular, y definen las composiciones de los medios de cultivo . [4] Un número limitado de factores de señalización, como factores de crecimiento o moléculas pequeñas , que controlan la diferenciación celular, se aplica de forma secuencial o combinatoria, en dosis y tiempos de exposición variables . [1] La diferenciación adecuada del tipo celular de interés se verifica mediante el análisis de marcadores específicos del tipo celular , el perfil de expresión génica y los ensayos funcionales. [1]
Métodos tempranos
- cocultivo con células estromales o células alimentadoras , y en sustratos de cultivo específicos:
support cells and matrices provide developmental-like environmental signals.[8]
- 3D cell aggregate formation, termed embryoid bodies (EBs): the aggregate aim at mimicking early embryonic development and instructing the cell differentiation.[1][5][8]
- culture in presence of fetal bovine serum, removal of pluripotency factors.
Current methodologies
Directed differentiation
This method consists in exposing the cells to specific signaling pathways modulators and manipulating cell culture conditions (environmental or exogenous) to mimick the natural sequence of developmental decisions to produce a given cell type/tissue.[1][8] A drawback of this approach is the necessity to have a good understanding of how the cell type of interest is formed.[1]
Direct reprogramming
This method, also known as transdifferentiation or direct conversion, consists in overexpressing one or several factors, usually transcription factors, introduced in the cells.[1] The starting material can be either pluripotent stem cells (PSCs), or either differentiated cell type such as fibroblasts. The principle was first demonstrated in 1987 with the myogenic factors MyoD.[9] A drawback of this approach is the introduction of foreign nucleic acid in the cells and the forced expression of transcription factors which effects are not fully understood.
Lineage/cell type-specific selection
This methods consists in selecting the cell type of interest, usually with antibiotic resistance. For this purpose, the source material cells are modified to contain antibiotic resistance cassette under a target cell type specific promoter.[10][11] Only cells committed to the lineage of interest is surviving the selection.
Aplicaciones
Directed differentiation provides a potentially unlimited and manipulable source of cell and tissues. Some applications are impaired by the immature phenotype of the pluripotent stem cells (PSCs)-derived cell type, which limits the physiological and functional studies possible.[6] Several application domains emerged:
Model system for basic science
For basic science, notably developmental biology and cell biology, PSC-derived cells allow to study at the molecular and cellular levels fundamental questions in vitro,[5] that would have been otherwise extremely difficult or impossible to study for technical and ethical reasons in vivo such as embryonic development of human. In particular, differentiating cells are amenable for quantitative and qualitative studies.[8] More complex processes can also be studied in vitro and formation of organoids, including cerebroids, optic cup and kidney have been described.
Drug discovery and toxicology
Cell types differentiated from pluripotent stem cells (PSCs) are being evaluated as preclinical in vitro models of Human diseases.[5] Human cell types in a dish provide an alternative to traditional preclinical assays using animal, human immortalized cells or primary cultures from biopsies, which have their limitations. Clinically-relevant cell types i.e. cell type affected in diseases are a major focus of research, this includes hepatocytes, Langerhans islet beta-cells,[12] cardiomyocytes and neurons. Drug screen are performed on miniaturized cell culture in multiwell-plates or on a chip.[6]
Disease modeling
PSCs-derived cells from patients are used in vitro to recreate specific pathologies.[6] The specific cell type affected in the pathology is at the base of the model. For example, motoneurons are used to study spinal muscular atrophy (SMA) and cardiomyocytes[2] are used to study arrythmia. This can allow for a better understanding of the pathogenesis and the development of new treatments through drug discovery.[6] Immature PSC-derived cell types can be matured in vitro by various strategies, such as in vitro ageing, to modelize age-related disease in vitro. Major diseases being modelized with PSCs-derived cells are amyotrophic lateral sclerosis (ALS), Alzheimer's (AD), Parkinson's (PD), fragile X syndrome (FXS), Huntington disease (HD), Down syndrome, Spinal muscular atrophy (SMA), muscular dystrophies,[13][14] cystic fibrosis, Long QT syndrome, and Type I diabetes.[6]
Regenerative medicine
The potentially unlimited source of cell and tissues may have direct application for tissue engineering, cell replacement and transplantation following acute injuries and reconstructive surgery.[2][5] These applications are limited to the cell types that can be differentiated efficiently and safely from human PSCs with the proper organogenesis.[1] Decellularized organs are also being used as tissue scaffold for organogenesis. Source material can be normal healthy cells from another donor (heterologous transplantation) or genetically corrected from the same patient (autologous). Concerns on patient safety have been raised due to the possibility of contaminating undifferentiated cells. The first clinical trial using hESC-derived cells was in 2011.[15] The first clinical trial using hiPSC-derived cells started in 2014 in Japan.[16]
Referencias
- ^ a b c d e f g h i j k Cohen DE, Melton D (2011). "Turning straw into gold: directing cell fate for regenerative medicine". Nature Reviews Genetics. 12 (4): 243–252. doi:10.1038/nrg2938. PMID 21386864.
- ^ a b c d Murry CE, Keller G (2008). "Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development". Cell. 132 (4): 661–680. doi:10.1016/j.cell.2008.02.008. PMID 18295582. Retrieved 2014-11-06.
- ^ Wichterle H, Lieberam I, Porter JA, Jessell TM (2002). "Directed differentiation of embryonic stem cells into motor neurons". Cell. 110 (3): 385–397. doi:10.1016/S0092-8674(02)00835-8. PMID 12176325.
- ^ a b c d e Spagnoli FM, Hemmati-Brivanlou A (2006). "Guiding embryonic stem cells towards differentiation: lessons from molecular embryology". Current Opinion in Genetics & Development. 16 (5): 469–475. doi:10.1016/j.gde.2006.08.004. PMID 16919445.
- ^ a b c d e f Keller G. "Embryonic stem cell differentiation: emergence of a new era in biology and medicine". genesdev.cshlp.org. PMID 15905405. Retrieved 2014-11-06. Cite journal requires
|journal=
(help) - ^ a b c d e f Sterneckert JL, Reinhardt P, Schöler HR (2014). "Investigating human disease using stem cell models". Nature Reviews Genetics. 15 (9): 625–639. doi:10.1038/nrg3764. PMID 25069490.
- ^ Jones-Villeneuve EM, McBurney MW, Rogers KA, Kalnins VI (1982). "Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells". The Journal of Cell Biology. The Rockefeller University Press. 94 (2): 253–262. doi:10.1083/jcb.94.2.253. PMC 2112882. PMID 7107698.
- ^ a b c d Nishikawa S, Jakt LM, Era T (2007). "Embryonic stem-cell culture as a tool for developmental cell biology". Nature Reviews Molecular Cell Biology. 8 (6): 502–507. doi:10.1038/nrm2189. PMID 17522593.
- ^ Davis RL, Weintraub H, Lassar AB (1987). "Expression of a single transfected cDNA converts fibroblasts to myoblasts". Cell. 51 (6): 987–1000. doi:10.1016/0092-8674(87)90585-X. PMID 3690668.
- ^ Marchetti S, Gimond C, Iljin K, Bourcier C, Alitalo K, Pouysségur J, Pagès G (2002). "Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo". Journal of Cell Science. 115 (Pt 10): 2075–2085. PMID 11973349.
- ^ Klug MG, Soonpaa MH, Koh GY, Field LJ (1996). "Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts". Journal of Clinical Investigation. 98 (1): 216–224. doi:10.1172/JCI118769. PMC 507419. PMID 8690796.
- ^ Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R (2001). "Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets". Science. 292 (5520): 1389–1394. doi:10.1126/science.1058866. PMID 11326082. S2CID 13025470.
- ^ Chal J, Oginuma M, Al Tanoury Z, Gobert B, Sumara O, Hick A, Bousson F, Zidouni Y, Mursch C, Moncuquet P, Tassy O, Vincent S, Miyanari A, Bera A, Garnier JM, Guevara G, Hestin M, Kennedy L, Hayashi S, Drayton B, Cherrier T, Gayraud-Morel B, Gussoni E, Relaix F, Tajbakhsh S, Pourquié O (August 2015). "Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy" (PDF). Nature Biotechnology. 33 (9): 962–9. doi:10.1038/nbt.3297. PMID 26237517.
- ^ Shelton M, Kocharyan A, Liu J, Skerjanc IS, Stanford WL (2016). "Robust generation and expansion of skeletal muscle progenitors and myocytes from human pluripotent stem cells". Methods. 101: 73–84. doi:10.1016/j.ymeth.2015.09.019. PMID 26404920.
- ^ "First test of human embryonic stem cell therapy in people discontinued - The Washington Post". washingtonpost.com. Retrieved 2014-11-06.
- ^ "Japanese team first to use iPS cells in bid to restore human sight | The Japan Times". japantimes.co.jp. Retrieved 2014-11-06.