The use of agarose microwells for scalable embryoid body formation and cardiac differentiation of human and murine pluripotent stem cells.

In most pluripotent stem cell differentiation protocols the formation of embryoid bodies (EBs) is an important step. Here we describe a rapid, straightforward soft lithography approach for the preparation of hydrophilic silicon masters from different templates and the subsequent production of patterned agarose-DMEM microwell surfaces for scalable well standardized stem cell aggregation and EB formation. The non-adhesive agarose microwell plates represent an accurate replication of the templates' topography and were used for aggregation of murine induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs). Direct microscopic assessment by time-lapse analysis demonstrated rapid formation of uniformly shaped EBs from murine iPSCs with similar or even more consistent results concerning size distribution and harvesting efficiency compared to the commonly used but time-consuming hanging drop technique. For human ESCs, homogenous aggregates were obtained after single cell inoculation on agarose microwells with efficient differentiation into the cardiac lineage using state-of-the-art protocols for directed differentiation via small molecules. With this soft lithography-based strategy, sufficient and reproducible numbers of stem cell-derived cardiomyocytes necessary for tissue engineering purposes can be realized in a highly controllable manner. Moreover, it might be useful for different cell types in any application that requires scalable and highly standardized aggregation.

Higher frequencies of BCRP+ cardiac resident cells in ischaemic human myocardium.

AIMS: Several cardiac resident progenitor cell types have been reported for the adult mammalian heart. Here we characterize their frequencies and distribution pattern in non-ischaemic human myocardial tissue and after ischaemic events. METHODS AND RESULTS: We obtained 55 biopsy samples from human atria and ventricles and used immunohistological analysis to investigate two cardiac cell types, characterized by the expression of breast cancer resistance protein (BCRP)/ABCG2 [for side population (SP) cells] or c-kit. Highest frequencies of BCRP+ cells were detected in the ischaemic right atria with a median of 5.40% (range: 2.48-11.1%) vs. 4.40% (1.79-7.75%) in the non-ischaemic right atria (P = 0.47). Significantly higher amounts were identified in ischaemic compared with non-ischaemic ventricles, viz. 5.44% (3.24-9.30%) vs. 0.74% (0-5.23%) (P = 0.016). Few numbers of BCRP+ cells co-expressed the cardiac markers titin, sarcomeric α-actinin, or Nkx2.5; no co-expression of BCRP and progenitor cell marker Sca-1 or pluripotency markers Oct-3/4, SSEA-3, and SSEA-4 was detected. C-kit+ cells displayed higher frequencies in ischaemic (ratio: 1:25 000 ± 2500 of cell counts) vs. non-ischaemic myocardium (1:105 000 ± 43 000). Breast cancer resistance protein+/c-kit+ cells were not identified. Following in vitro differentiation, BCRP+ cells isolated from human heart biopsy samples (n = 6) showed expression of cardiac troponin T and α-myosin heavy-chain, but no full differentiation into functional beating cardiomyocytes was observed. CONCLUSION: We were able to demonstrate that BCRP+/CD31- cells are more abundant in the heart than their c-kit+ counterparts. In the non-ischaemic hearts, they are preferentially located in the atria. Following ischaemia, their numbers are elevated significantly. Our data might provide a valuable snapshot at potential progenitor cells after acute ischaemia in vivo, and mapping of these easily accessible cells may influence future cell therapeutic strategies.

Murine and human pluripotent stem cell-derived cardiac bodies form contractile myocardial tissue in vitro.

AIMS: We explored the use of highly purified murine and human pluripotent stem cell (PSC)-derived cardiomyocytes (CMs) to generate functional bioartificial cardiac tissue (BCT) and investigated the role of fibroblasts, ascorbic acid (AA), and mechanical stimuli on tissue formation, maturation, and functionality. METHODS AND RESULTS: Murine and human embryonic/induced PSC-derived CMs were genetically enriched to generate three-dimensional CM aggregates, termed cardiac bodies (CBs). Addressing the critical limitation of major CM loss after single-cell dissociation, non-dissociated CBs were used for BCT generation, which resulted in a structurally and functionally homogenous syncytium. Continuous in situ characterization of BCTs, for 21 days, revealed that three critical factors cooperatively improve BCT formation and function: both (i) addition of fibroblasts and (ii) ascorbic acid supplementation support extracellular matrix remodelling and CB fusion, and (iii) increasing static stretch supports sarcomere alignment and CM coupling. All factors together considerably enhanced the contractility of murine and human BCTs, leading to a so far unparalleled active tension of 4.4 mN/mm(2) in human BCTs using optimized conditions. Finally, advanced protocols were implemented for the generation of human PSC-derived cardiac tissue using a defined animal-free matrix composition. CONCLUSION: BCT with contractile forces comparable with native myocardium can be generated from enriched, PSC-derived CMs, based on a novel concept of tissue formation from non-dissociated cardiac cell aggregates. In combination with the successful generation of tissue using a defined animal-free matrix, this represents a major step towards clinical applicability of stem cell-based heart tissue for myocardial repair.

Rhesus monkey cardiosphere-derived cells for myocardial restoration.

BACKGROUND AIMS: Cardiosphere-derived cells (CDC) have been proposed as a promising myocardial stem cell source for cardiac repair. They have been isolated from human, porcine and rodent cardiac biopsies. However, their usefulness for myocardial restoration remains controversial. We aimed to determine the survival, differentiation and functional effects of Rhesus monkey CDC (RhCDC) in a mouse model of myocardial infarction. METHODS: RhCDC were isolated and characterized by flow cytometry and reverse transcriptase (RT)-polymerase chain reaction (PCR) and compared with human CDC. They were injected intramyocardially into severe combined immune deficiency (SCID) beige mice after ligature of the left anterior descending artery (LAD). Phosphate-buffered saline (PBS) served as placebo. Medium treatment alone was used to distinguish between cellular and non-cellular effects. Animals were divided into a non-infarcted control group (n = 7), infarct control groups (n = 24), medium-treated infarct groups (n = 35) and RhCDC-treated infarct groups (n = 33). Follow-up was either 1 or 4 weeks. LV function was assessed by pressure-volume loop analysis. Differentiation was analyzed by immunhistochemical profiling and RT-PCR. RESULTS: Proliferating RhCDC grafts were detected after transplantation in an acute infarct model. RhCDC as well as medium treatment protected myocardium within the infarct area and improved LV function. RhCDC had a superior regenerative effect than medium alone. CONCLUSIONS: For the first time, RhCDC have been used for the restoration of infarcted myocardium. RhCDC proliferated in vivo and positively influenced myocardial remodeling. This effect could be mimicked by treatment with unconditioned medium alone, emphasizing a non-cellular paracrine therapeutic mechanism. However, as a robust cardiac stem cell source, CDC might be useful to evoke prolonged paracrine actions in cardiac stem cell therapy.