RESUMEN
BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former. CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.
Asunto(s)
Diferenciación Celular , Ácido Hialurónico , Células Madre Mesenquimatosas , Células Madre Pluripotentes , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Ácido Hialurónico/farmacología , Ácido Hialurónico/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Medio de Cultivo Libre de Suero/farmacología , Linaje de la Célula , Células Cultivadas , Técnicas de Cultivo de Célula/métodos , Técnicas de CocultivoRESUMEN
Disease-relevant human induced pluripotent stem cells (iPSCs) are generated worldwide for research purposes; however, without robust and practical ethical, legal, and quality standards, there is a high risk that their true potential will not be realized. Best practices for tissue procurement, iPSC reprogramming, day-to-day cultivation, quality control, and data management aligned with an ethical and legal framework must be included into daily operations to ensure their promise is maximized. Here we discuss key learning experiences from 7 years of operating the European Bank for induced Pluripotent Stem Cells (EBiSC) and recommend how to incorporate solutions into a daily management framework.
Asunto(s)
Bancos de Muestras Biológicas/estadística & datos numéricos , Reprogramación Celular/genética , Criopreservación/métodos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Bancos de Muestras Biológicas/ética , Bancos de Muestras Biológicas/normas , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/normas , Diferenciación Celular/genética , Línea Celular , Europa (Continente) , Humanos , Control de CalidadRESUMEN
We report the first investigation into the bioprinting of human induced pluripotent stem cells (hiPSCs), their response to a valve-based printing process as well as their post-printing differentiation into hepatocyte-like cells (HLCs). HLCs differentiated from both hiPSCs and human embryonic stem cells (hESCs) sources were bioprinted and examined for the presence of hepatic markers to further validate the compatibility of the valve-based bioprinting process with fragile cell transfer. Examined cells were positive for nuclear factor 4 alpha and were demonstrated to secrete albumin and have morphology that was also found to be similar to that of hepatocytes. Both hESC and hiPSC lines were tested for post-printing viability and pluripotency and were found to have negligible difference in terms of viability and pluripotency between the printed and non-printed cells. hESC-derived HLCs were 3D printed using alginate hydrogel matrix and tested for viability and albumin secretion during the remaining differentiation and were found to be hepatic in nature. 3D printed with 40-layer of HLC-containing alginate structures reached peak albumin secretion at day 21 of the differentiation protocol. This work demonstrates that the valve-based printing process is gentle enough to print human pluripotent stem cells (hPSCs) (both hESCs and hiPSCs) while either maintaining their pluripotency or directing their differentiation into specific lineages. The ability to bioprint hPSCs will pave the way for producing organs or tissues on demand from patient specific cells which could be used for animal-free drug development and personalized medicine.
Asunto(s)
Bioimpresión/métodos , Diferenciación Celular , Hepatocitos/citología , Hígado/citología , Células Madre Pluripotentes/citología , Impresión Tridimensional , Biomarcadores/metabolismo , Bioimpresión/instrumentación , Diferenciación Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Hepatocitos/efectos de los fármacos , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Microscopía Fluorescente , Presión , Reproducibilidad de los Resultados , EsterilizaciónRESUMEN
In recent years, the use of a simple inkjet technology for cell printing has triggered tremendous interest and established the field of biofabrication. A key challenge has been the development of printing processes which are both controllable and less harmful, in order to preserve cell and tissue viability and functions. Here, we report on the development of a valve-based cell printer that has been validated to print highly viable cells in programmable patterns from two different bio-inks with independent control of the volume of each droplet (with a lower limit of 2 nL or fewer than five cells per droplet). Human ESCs were used to make spheroids by overprinting two opposing gradients of bio-ink; one of hESCs in medium and the other of medium alone. The resulting array of uniform sized droplets with a gradient of cell concentrations was inverted to allow cells to aggregate and form spheroids via gravity. The resulting aggregates have controllable and repeatable sizes, and consequently they can be made to order for specific applications. Spheroids with between 5 and 140 dissociated cells resulted in spheroids of 0.25-0.6 mm diameter. This work demonstrates that the valve-based printing process is gentle enough to maintain stem cell viability, accurate enough to produce spheroids of uniform size, and that printed cells maintain their pluripotency. This study includes the first analysis of the response of human embryonic stem cells to the printing process using this valve-based printing setup.
Asunto(s)
Bioimpresión/métodos , Células Madre Embrionarias/citología , Ingeniería de Tejidos/instrumentación , Andamios del Tejido/química , Proliferación Celular , Supervivencia Celular , Células Madre Embrionarias/química , Humanos , Tamaño de la PartículaRESUMEN
The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.
Asunto(s)
Células Madre Embrionarias/trasplante , Obtención de Tejidos y Órganos , Animales , Línea Celular , Selección de Donante/ética , Células Madre Embrionarias/citología , Fertilización In Vitro , Humanos , Donadores Vivos/ética , Donadores Vivos/legislación & jurisprudencia , Manejo de Especímenes , Investigación con Células Madre/ética , Investigación con Células Madre/legislación & jurisprudencia , Trasplante de Células Madre/ética , Trasplante de Células Madre/normas , Bancos de Tejidos , Obtención de Tejidos y Órganos/ética , Obtención de Tejidos y Órganos/legislación & jurisprudenciaRESUMEN
Scotland is shaping up as Europe's 'go-to' place to translate cell therapy research from the laboratory into the clinic. While continuing to punch above its weight in fundamental research, the resources for translating research to clinical application are perhaps its main attraction for the international stem cell research community.