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Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

Fuerstenau-Sharp M, Zimmermann ME, Stark K, Jentsch N, Klingenstein M, Drzymalski M, Wagner S, Maier LS, Hehr U, Baessler A, Fischer M, Hengstenberg C - PLoS ONE (2015)

Bottom Line: The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V).Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes.It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Clinic for Internal Medicine II, University Hospital Regensburg, Regensburg, Germany.

ABSTRACT
Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

No MeSH data available.


Action potentials characteristics of iPS cell-derived cardiomyocytes.A: Left panel shows exemplary original traces of APs of spontaneously contracting cardiomyocytes. Distinct AP morphologies representing ventricular-, atrial-, and nodal-like cardiomyoctes were discriminated. Right panel shows the proportional distribution. B: Original traces of APs induced by current injection (current clamp) at various frequencies. Interestingly, iPS cell-derived cardiomyocytes responded to stimulation rates as high as 3 Hz. Mean data for AP duration at 30% and 90% repolarization (APD30 and 90, respectively) showed a rate-dependent shortening in steady-state AP duration.
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pone.0126596.g004: Action potentials characteristics of iPS cell-derived cardiomyocytes.A: Left panel shows exemplary original traces of APs of spontaneously contracting cardiomyocytes. Distinct AP morphologies representing ventricular-, atrial-, and nodal-like cardiomyoctes were discriminated. Right panel shows the proportional distribution. B: Original traces of APs induced by current injection (current clamp) at various frequencies. Interestingly, iPS cell-derived cardiomyocytes responded to stimulation rates as high as 3 Hz. Mean data for AP duration at 30% and 90% repolarization (APD30 and 90, respectively) showed a rate-dependent shortening in steady-state AP duration.

Mentions: In order to investigate the electrical activity of differentiated cardiomyocytes, whole-cell patch clamp technique was performed. Fig 4A shows original traces of spontaneous actions potentials (APs) recorded in current clamp mode. In the population of cells investigated (n = 8), 50% of the cells displayed ventricular-like APs, while 37.5% had atrial-like AP and only 12.5% showed action potentials that are consistent with nodal cardiomyocytes. Detailed analysis of resting membrane potential (RMP), action potential amplitude (APA), maximal AP upstroke velocity (APA) and action potential duration at 30%, 50%, 80% and 90% repolarization (APD 30, 50, 80 and 90) are shown in Table 2. For a subgroup of investigated cardiomyocytes, APs were elicited using square current pulses at variable frequency (0.5–3 Hz). As shown in Fig 4B, a frequency dependent shortening of the steady-state AP duration could be observed in the iPS cell-derived cardiomyocytes. The underlying mechanism of rate-dependent shortening of AP duration includes an increase in slow delayed rectifier potassium current, IKs, [34] and increased Na-Ca exchanger current [35]. This suggests that the investigated cardiomyocytes may express a functional IKs and Na-Ca exchanger current.


Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

Fuerstenau-Sharp M, Zimmermann ME, Stark K, Jentsch N, Klingenstein M, Drzymalski M, Wagner S, Maier LS, Hehr U, Baessler A, Fischer M, Hengstenberg C - PLoS ONE (2015)

Action potentials characteristics of iPS cell-derived cardiomyocytes.A: Left panel shows exemplary original traces of APs of spontaneously contracting cardiomyocytes. Distinct AP morphologies representing ventricular-, atrial-, and nodal-like cardiomyoctes were discriminated. Right panel shows the proportional distribution. B: Original traces of APs induced by current injection (current clamp) at various frequencies. Interestingly, iPS cell-derived cardiomyocytes responded to stimulation rates as high as 3 Hz. Mean data for AP duration at 30% and 90% repolarization (APD30 and 90, respectively) showed a rate-dependent shortening in steady-state AP duration.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430251&req=5

pone.0126596.g004: Action potentials characteristics of iPS cell-derived cardiomyocytes.A: Left panel shows exemplary original traces of APs of spontaneously contracting cardiomyocytes. Distinct AP morphologies representing ventricular-, atrial-, and nodal-like cardiomyoctes were discriminated. Right panel shows the proportional distribution. B: Original traces of APs induced by current injection (current clamp) at various frequencies. Interestingly, iPS cell-derived cardiomyocytes responded to stimulation rates as high as 3 Hz. Mean data for AP duration at 30% and 90% repolarization (APD30 and 90, respectively) showed a rate-dependent shortening in steady-state AP duration.
Mentions: In order to investigate the electrical activity of differentiated cardiomyocytes, whole-cell patch clamp technique was performed. Fig 4A shows original traces of spontaneous actions potentials (APs) recorded in current clamp mode. In the population of cells investigated (n = 8), 50% of the cells displayed ventricular-like APs, while 37.5% had atrial-like AP and only 12.5% showed action potentials that are consistent with nodal cardiomyocytes. Detailed analysis of resting membrane potential (RMP), action potential amplitude (APA), maximal AP upstroke velocity (APA) and action potential duration at 30%, 50%, 80% and 90% repolarization (APD 30, 50, 80 and 90) are shown in Table 2. For a subgroup of investigated cardiomyocytes, APs were elicited using square current pulses at variable frequency (0.5–3 Hz). As shown in Fig 4B, a frequency dependent shortening of the steady-state AP duration could be observed in the iPS cell-derived cardiomyocytes. The underlying mechanism of rate-dependent shortening of AP duration includes an increase in slow delayed rectifier potassium current, IKs, [34] and increased Na-Ca exchanger current [35]. This suggests that the investigated cardiomyocytes may express a functional IKs and Na-Ca exchanger current.

Bottom Line: The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V).Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes.It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Clinic for Internal Medicine II, University Hospital Regensburg, Regensburg, Germany.

ABSTRACT
Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

No MeSH data available.