Вывод из высокоочищенных кардиомиоцитов из человеческих индуцированных плюрипотентных стволовых клеток с использованием малой молекулы-модулированный Дифференциация и последующих глюкозы Голод

Summary

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Abstract

Клеток получают кардиомиоциты плюрипотентные стволовые вызванных деятельностью человека (hiPSC-CMS) стали важным источником клеток для решения проблемы нехватки первичных кардиомиоцитов, доступных для фундаментальных исследований и трансляционных приложений. Чтобы дифференцировать hiPSCs в кардиомиоциты, различные протоколы, включая эмбриоидном тела (EB) основе дифференциации и индукции фактора роста были разработаны. Тем не менее, эти протоколы неэффективны и сильно варьирует в их способности генерировать очищенные кардиомиоциты. В последнее время, малая молекула на основе протокола использованием модуляции сигналов Wnt / β-катенин было показано, чтобы способствовать сердечной дифференциации с высокой эффективностью. С помощью этого протокола, более чем на 50% -60% дифференцированных клеток были сердечный тропонин-положительных кардиомиоциты последовательно наблюдалось. Для дальнейшего повышения чистоты кардиомиоцитов, дифференцированные клетки подвергали голоданию в глюкозу, чтобы специально исключить некардиомиоциты на основе метаболического разностис между кардиомиоцитов и некардиомиоциты. Используя эту стратегию выбора, мы последовательно получили увеличение более чем на 30% в соотношении кардиомиоцитов в некардиомиоциты в популяции дифференцированных клеток. Эти особо чистые кардиомиоциты должны повысить надежность результатов человеческой IPSC основе в пробирке исследования моделирования заболеваний и скрининга лекарственных средств анализов.

Introduction

Primary human cardiomyocytes are difficult to obtain because of the requirement for invasive cardiac biopsies, difficulty in dissociating to single cells, and because of poor long-term cell survival in culture. Given this lack of primary human cardiomyocytes, patient-specific human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology has been regarded as a powerful alternative cardiomyocyte source for basic research as well as clinical and translational applications such as disease modeling and drug discovery¹. Early efforts in differentiating pluripotent stem cells into cardiomyocytes employed differentiation protocols using embryoid bodies (EBs), but this method is inefficient in producing cardiomyocytes because often less than 25% of cells in an EB are beating cardiomyocytes^2,3. Comparatively, a monolayer-based differentiation protocol using the cytokines activin A and BMP4 displayed a higher efficiency than EBs, but this protocol is still relatively inefficient, requires expensive growth factors, and only functions in a limited number of human pluripotent stem cell lines⁴. Recently, a highly efficient, hiPSC monolayer-based cardiomyocyte differentiation protocol was developed by modulating Wnt/β-Catenin signaling⁵. These hiPSC-CMs express cardiac troponin T and alpha actinin, two sarcomeric proteins that are standard markers of cardiomyocytes⁶. The protocol describe here is an adaptation of this small molecule-based, feeder cell-free, monolayer differentiation method^5,7. We are able to obtain beating cardiomyocytes from hiPSCs after 7-10 days (Figure 1). However, following a cardiomyocyte differentiation resulting in 50% beating cells, immunostaining consistently shows the existence of a population of non-cardiomyocytes that are negative for cardiomyocyte-specific markers such as cardiac-specific troponin T and alpha-actinin. To further purify cardiomyocytes and eliminate non-cardiomyocytes, heterogeneous differentiated cell populations were subjected to glucose starvation by treating them with an extremely low glucose culture medium for multiple days (Figure 2). This treatment selectively eliminates non-cardiomyocytes due to the ability of cardiomyocytes, but not non-cardiomyocytes, to metabolize lactate as the primary energy source in order to survive in a low glucose environment⁸. After this purification step, a 40% increase in the ratio of cardiomyocytes to non-cardiomyocytes is observed, (Figure 3, Figure 4) and these cells can be used for downstream gene expression analysis, disease modeling, and drug screening assays.

Protocol

NOTE: Vendor information for all reagents used in this protocol has been listed in Table 1 and Materials List. All solutions and equipment coming into contact with cells must be sterile, and aseptic technique should be used accordingly. Perform all culture incubations in a humidified 37 °C, 5% CO2 incubator unless otherwise specified. In this protocol, all differentiations are performed in 6-well plates, in which the hiPSCs are seeded. Following differentiation and purifi…

Representative Results

The morphological changes during hiPSC differentiation. The hiPSC cultured in feeder-free plates grew as flat, two-dimensional colonies. Upon reaching around 85% confluency, hiPSCs were treated with 6 µM CHIR for differentiation (Figure 1A). Substantial amounts of cell death, a normal and common phenomenon, were observed after 24 hr of CHIR treatment. After two days of CHIR treatment, the hiPSCs continued to differentiate towards a mesodermal fate. In com…

Discussion

Obtaining a large amount of highly purified hiPSC-derived cardiomyocytes is critical for basic cardiac research as well as clinical and translational applications. Cardiac differentiation protocols have undergone tremendous improvements in recent years, transitioning from embryoid body-based methods utilizing cardiogenic growth factors², to matrix sandwich methods¹², and finally to small molecule-modulated and monolayer-based methods⁵. Of the aforementioned protocols, the protocol describ…

Materials

Name	Company	Catalog number
Matrigel (9-12 mg/mL)	BD Biosciences	354277
RPMI media	Invitrogen	11835055
Glucose free RPMI media	Invitrogen	11879-020
B27 Minus Insulin	Invitrogen	A1895601
B27 Supplement (w/ insulin)	Invitrogen	17504-044
Pen-strep antibiotic	Invitrogen	15140122
Fetal bovine serum	BenchMark	100-106
DMSO	Sigma	D-2650
ROCK inhibitor Y-27632	EMD Millipore	688000
CHIR99021	Thermo Fisher	508306
IWR1	Sigma	I0161
EDTA	Invitrogen	15575-020
Accutase	Millipore	SCR005
Cell lifter	Fisher	08-100-240
Cryovial	Fisher (NUNC tubes)	375418
TrypLE Select Enzyme	Invitrogen	12563-011