Question

written assignment for a research article

Title: Distinct Metabolic Flow Enables Large-Scale Purification of Mouse and Human Pluripotent Stem Cell-Derived Cardiomyocytes

Link: https://www.sciencedirect.com/science/article/pii/S1934590912005796

Suggested Flow:

a). Background and rationale (Please describe the background of research)

b). novelty and significance

c). method: (Please describe the methodologies this article employed)

d). summary of results: (Please describe the major findings)

e). discussion: (Please describe the discussion with your own words, not simply rewrite the article)

f). limitation: (Please describe the limitation of research)

g). future implication: (Please describe any future direction you can suggest based on this article)

I have completed this assignment but I want to compare my work with an expert

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Answer 1

Background :

Heart disease is a common and deadly disease, and heartregenerative therapy is a promising therapeutic strategy for some patients (Passier et al., 2008). Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are potential sources for production of therapeutic cardiomyocytes.

Procedures involving density-gradient centrifugation, genetic modification and nongenetic methods that use a
mitochondrial dye or antibodies to specific cell-surface markers have been established for cardiomyocyte enrichment. However, none of these methods are ideal for the therapeutic application of PSC-derived cardiomyocytes because of drawbacks including insufficient purity, genotoxicity, and the use of luorescenceactivated
cell sorting (FACS) and/or antibodies.

Novelty

In this study, they have took advantage of the unique metabolic properties of cardiomyocytes to develop an efficient and noninvasive environmental approach for their purification from PSC cultures.

This method scale is close to the capacity of commercially available automatic large-scale culture systems and suggests that combining more sophisticated differentiation methods with our lactate method could facilitate realistic application of PSC-derived cardiomyocytes to human therapy.

A nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells.

Methods:

Invivo and invitro studies were done

Fluorescence microscopy or confocal microscopy

Flow cytometry

Real time PCR

Mass spectroscpopy and capillary electrophoresis

Action-Potential recordings using Microelectrodes

Field-Potential recordings using the MEA System

Teratoma Formation, Colony-Formation Assay, ATP Measurement, [14C]-Labeled Lactate Uptake, EdU Incorporation Assay

for Statistical analysis SPSS software was used

Summary of results

The results for cardiomyocytes revealed markedly higher expression of genes encoding enzymes involved in the
TCA cycle than the undifferentiated ESCs and, in turn, lower expression of genes involved in the pentose phosphate, amino acid synthesis, and lipid synthesis pathways.

They have compared the [14C]-lactate uptake activity of neonatal rat and human ESCderived cardiomyocytes, ESCs, MEFs, and noncontracting EBs, and found that both cardiomyocyte populations showed higher levels of activity than the other cells.

Lactate supplementation has the potential to cause acidification either intracellularly or in the medium, which could lead to cellular damage.

Counting of immunocytochemically a-actinin-positive cells after 2, 4, 6, and 8 days revealed that the purified cardiomyocytes could proliferate up to 2.5-fold in 8 days, and the fraction of multinuclear cardiomyocytes increased over time compared with the second day.

Discussion

They have reported a simple medium-exchanging procedure that enabled cardiomyocyte purification of up to 99% from a cell source comprising only 10% cardiomyocytes, with an estimated recovery of cardiomyocytes of 74.4 ± 12.1%, based on direct cell count before and after purification.

Then have eliminated the possibility that lactate supplementation caused toxic extracellular or intracellular acidification. Therefore, activation of the retrospective glycolytic pathway may accelerate a catastrophic balance of ATP supply and demand in ESCs, whereas cardiomyocytes can maintain cellular ATP homeostasis by producing
more ATP via a highly active OXPHOS mechanism.

They have obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation.