BACKGROUND: Human embryonic stem cells (hESCs) have the unique ability to differentiate into any cell type in the human body and to proliferate indefinitely. Cell therapies involving hESC have shown very promising results for the treatment of certain diseases and confirmed the safety of hESC-derived cells for humans. They are used in cell therapy, mainly in targeted therapy of diseases that are currently incurable. OBJECTIVES: The aim of this study was the derivation of clinical-grade hESCs usable in drug development, non-native medicine and cell therapy. MATERIAL AND METHODS: Embryos were thawed, cultivated to the blastocyst stage if necessary, and assisted hatching was subsequently performed. Embryoblasts were mechanically isolated using narrow needles. Each line was kept as a separate batch. The derived hESCs were cultured under hypoxic culture conditions (5% O2, 5% CO2, 37°C) in a NutriStem® hPSC XF Medium with a daily medium change. RESULTS: From January 2018 to July 2020, 138 selected clients were asked for consent to donate embryos, of whom 52 did not respond, 19 terminated the storage of their embryos and 29 extended the storage. Only 38 clients (27.5%) agreed to donate embryos for the derivation of hESCs. At the same time, personal communication with clients took place and another 17 embryo donors were recruited. A total of 160 embryos from 55 donors aged 26-42 years were collected. The embryos were frozen at the blastocyst (33.1%) or morula (46.3%) stage. After the preparation of 64 embryos, embryoblasts were isolated and cultured. Finally, 7 hESC lines were obtained, 4 research-grade and 3 clinical-grade, the first in the Czech Republic. CONCLUSIONS: We established a current good manufacturing practice (cGMP)-defined xeno-free and feeder-free system for the derivation, culture and banking of clinical-grade hESC lines that are suitable for preclinical and clinical trials. The quality control testing with criteria concerning sterility, safety and characterization according to cGMP ensured the clinical-grade quality of hESC lines.
- MeSH
- buněčné linie MeSH
- embryo savčí MeSH
- embryonální kmenové buňky * MeSH
- kvalita života * MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Human embryonic stem cells (hESCs) have unique abilities that enable their use in cell therapy, disease modeling, and drug development. Their derivation is usually performed using a feeder layer, which is undefined and can potentially cause a contamination by xeno components, therefore there is a tendency to replace feeders with xeno-free defined substrates in recent years. Three hESC lines were successfully derived on the vitronectin with a truncated N-terminus (VTN-N) in combination with E-cadherin in xeno-free conditions for the first time, and their undifferentiated state, hESC morphology, and standard karyotypes together with their potential to differentiate into three germ layers were confirmed. These results support the conclusion that the VTN-N/E-cadherin is a suitable substrate for the xeno-free derivation of hESCs and can be used for the derivation of hESCs according to good manufacturing practices.
- MeSH
- buněčná a tkáňová terapie MeSH
- kadheriny genetika MeSH
- lidé MeSH
- lidské embryonální kmenové buňky * MeSH
- obchod MeSH
- vitronektin MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Human embryonic stem cells (hESCs) are increasingly used in clinical trials as they can change the outcome of treatment for many human diseases. They are used as a starting material for further differentiation into specific cell types and to achieve the desirable result of the cell therapy; thus, the quality of hESCs has to be taken into account. Therefore, current good manufacturing practice (cGMP) has to be implemented in the transport of embryos, derivation of inner cell mass to xeno-free, feeder-free and defined hESC culture, and cell freezing. The in-depth characterization of hESC lines focused on safety, pluripotency, differentiation potential and genetic background has to complement this process. In this paper, we show the derivation of three clinical-grade hESC lines, MUCG01, MUCG02, and MUCG03, following these criteria. We developed and validated the system for the manufacture of xeno-free and feeder-free clinical-grade hESC lines that present high-quality starting material suitable for cell therapy according to cGMP.
Cíl práce: Práce se zabývá klinickou částí výzkumu lidských embryonálních kmenových buněk (hESC). Cílem projektu je vznik somatických buněčných typů použitelných ve vývoji léčiv, regenerativní medicíně a buněčné terapii. Výhledem je umožnit cílenou terapii dosud nevyléčitelné nemoci. Pluripotentní hESC mají neomezenou kapacitu pro sebeobnovu. Této vlastnosti se využívá v terapii, kdy jsou z hESC vytvořeny chybějící nebo poškozené buňky v lidském těle. Je zájem vytvořit hESC linie v klinické kvalitě, použitelné v předklinických a klinických studiích. Metodika: Vytvoření hESC musí respektovat legislativu ČR a EU. Podmínkou bylo vypracovat informovaný souhlas obou dárců pro darovaná vyřazená embrya, která nejsou vhodná pro léčbu oplodněním in vitro dle směrnice 2004/23/ES. Centrum asistované reprodukce (CAR) FN Brno se podílí na odběru oocytů, kultivaci a kryokonzervaci embryí, komunikaci s klienty a zajišťování informovaných souhlasů dárců embryí. Byl vypracován předávací protokol a metodika předávání rozmrazených embryí s originálním číselným kódem. Před předáním embryí na spoluautorské pracoviště – Centrum buněčného a tkáňového inženýrství (CTEF) ICRC FN u sv. Anny – je provedeno jejich rozmrazení, v případě potřeby dokultivování do stadia blastocysty, a následně je proveden asistovaný hatching. Výsledky: V období leden 2018 až červenec 2020 bylo obesláno 138 vybraných vhodných klientek na dárcovství, z nichž 52 nereagovalo, 19 ukončilo a 29 prodloužilo skladování embryí. Pouze 38 klientek, tj. 27,5 %, souhlasilo s jejich využitím na přípravu hESC. Ve stejném období probíhala osobní komunikace s vhodnými klienty CAR a bylo získáno dalších 17 dárců embryí. Celkem bylo získáno 160 embryí od 55 dárkyň ve věku 26–42 let. Nejčastěji byla embrya zamrazena ve stadiu blastocysty (53 embrií – 33,1 %) a moruly (74 embrií – 46,3 %). Z 29 geneticky vyšetřených embryí je 5 euploidních (17,2 %), 2 mozaiky a 22 aneuploidních nebo s translokací či přenašečů s monogenní vadou. Závěr: Byl vypracován a Etickou komisí LF MU a FN Brno schválen informovaný souhlas, bylo vybráno a zajištěno 160 darovaných embryí. Je vypracován předávací protokol a metodika předávání. Plán předávání rozmrazených anonymizovaných embryí zahrnuje cca 5 rozmrazených blastocyst týdně s provedeným asistovaným hatchingem. Po předání embryí na CTEF probíhá izolace embryoblastu s následnou kultivací. Ustanovené buněčné linie hESC musí splnit specifikovaná kritéria bezpečnosti, stability a pluripotence. Věříme, že v souladu s plánem projektu získáme nejméně tři linie hESC v klinické kvalitě, poprvé vytvořené v ČR, respektující požadavky na léčivé přípravky Advanced Medicinal Therapy Products (AMTP).
Objective: The work deals with a clinical part of human embryonic stem cell (hESC) research. The aim of the project is the differentiation of somatic cell types, useful in drug development, regenerative medicine and cell therapy. The aim of this work is to enable targeted therapy of yet incurable diseases. The pluripotent hESCs have unlimited self-renewal capacity. This ability is used in therapy to create missing or damaged cells in the human body. It is of interest to develop clinical-grade hESC lines useful in preclinical and clinical studies. Methods: The derivation of the hESC must respect the legislation of the Czech Republic and the EU. The aim was to develop an informed consent of both donors for donated discarded embryos that are not suitable for treatment by in vitro fertilization according to Directive 2004/23/EC. The FNB‘s Center for Assisted Reproduction (CAR) participates in oocyte collection, cultivation and cryopreservation of embryos, communication with clients and ensuring the informed consent of embryo donors. A transport protocol and a methodology for handing over the thawed embryos with the original numerical code were developed. Before the embryos are handed over to the ICRC co-author‘s workplace (CTEF), they are thawed and, if necessary, recultivated to the blastocyst stage; afterwards, assisted hatching is performed. Results: In the period from January 2018 to July 2020, 138 selected suitable clients were asked for donations, with 52 not responding, 19 terminating and 29 extending the embryo storage. Only 38 clients, i.e. 27.5%, agreed with the usage of their embryos for the preparation of hESCs. In the same period, personal communication with suitable CAR clients took place and another 17 embryo donors were obtained. A total of 160 embryos were obtained from 55 donors aged 26 to 42 years. The embryos were most often frozen in the blastocyst (53 embryos – 33.1%) and morula (74 embryos – 46.3%) stages. Of the 29 genetically examined embryos, only 5 are euploid (17.2%), 2 are mosaic and 22 are aneuploid or with translocations or carriers with a monogenic defect. Conclusion: We have an informed consent prepared and approved by the Ethics Committee of the Masaryk University and the University Hospital Brno; 160 donated embryos have been selected and secured. A transport protocol and handover methodology are developed. The plan for the transfer of thawed anonymized embryos in the first phase, October – December 2020, includes approximately 5 thawed blastocysts per week with assisted hatching. After their transfer to the CTEF, the embryoblast will be isolated with subsequent cultivation. The established hESCs must meet the specified criteria of safety, stability and pluripotency. We believe that, in accordance with the project plan, we will obtain at least 3 clinical-grade hESC lines, the first created in the Czech Republic, respecting the requirements for Advanced Medicinal Therapy Products (AMTP).
- Klíčová slova
- výběr embryí,
- MeSH
- blastocysta MeSH
- buněčná a tkáňová terapie MeSH
- embryoblast MeSH
- lidé MeSH
- lidské embryonální kmenové buňky MeSH
- nakládání s embryem * MeSH
- vitrifikace MeSH
- získávání tkání a orgánů MeSH
- Check Tag
- lidé MeSH
Human pluripotent stem cells have the potential to change the way in which human diseases are cured. Clinical-grade human embryonic stem cells and human induced pluripotent stem cells have to be created according to current good manufacturing practices and regulations. Quality and safety must be of the highest importance when humans' lives are at stake. With the rising number of clinical trials, there is a need for a consensus on hPSCs characterization. Here, we summarize mandatory and 'for information only' characterization methods with release criteria for the establishment of clinical-grade hPSC lines.
