Leonhard Linta, Marianne Stockmann, Qiong Lin, André Lechel, Christian Proepper, Tobias M. Boeckers, Alexander Kleger, Stefan Liebau, "Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny", Stem Cells International, vol. 2013, Article ID 784629, 25 pages, 2013. https://doi.org/10.1155/2013/784629
Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny
Leonhard Linta,1Marianne Stockmann,1Qiong Lin,2André Lechel,3Christian Proepper,1Tobias M. Boeckers,1Alexander Kleger,3 and Stefan Liebau1
2Institute for Biomedical Engineering, Department of Cell Biology, RWTH Aachen, Pauwelstrasse 30, 52074 Aachen, Germany
3Department of Internal Medicine I, Ulm University, Albert-Einstein Allee 11, 89081 Ulm, Germany
Academic Editor: Michael Levin
Received31 Jan 2013
Accepted06 Mar 2013
Published15 Apr 2013
Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms.
Ion channels are comprised of a large variety of differing families of pore proteins. Initially, ion channels were mostly known for their role in the nervous system where they play a crucial role in the signal transmission over neurites and synapses. But in fact they are involved in numerous other cellular processes including cell size regulation, muscle contractions, immune system activation , or hormone release . Distinct ion channels are furthermore recognized to be of high importance for excitable cells of the heart: cardiomyocytes of the working myocardium as well as cells of the cardiac conduction system. In the heart, specific ion channels are responsible, for example, for the regulated generation of action potentials and for cardiac muscle contraction strength and time . Additionally, ion channels play an important role in several differentiation and maturation processes [4–6]. The presented study aims to take a closer look at ion channel expression in human-induced pluripotent stem cells (hiPSCs) to give a start point for further analyses of their distinct roles at an early developmental cell state and differentiation processes.
hiPSCs are generated from somatic cells by timed overexpression of specific transcription factors and strongly resemble pluripotent embryonic stem cells [7, 8]. Pluripotency is amongst others defined by the ability to differentiate into cells of all three germ layers and unlimited symmetrical cell division. This cell system is widely utilized for studies investigating developmental processes or disease mechanisms [9, 10]. Although it has become clear that differentiation processes influence ion channel expression , the distinct role of ion channels during these processes is so far only poorly understood. Nevertheless, it became evident that certain ion channels play a pivotal role in stem cell biology, including cell fate determination, cell cycle regulation, or cytoskeletal reorganization [6, 12–15].
The possibilities of iPSCs include the generation of individual or patient-specific-pluripotent cells, which can be subsequently differentiated into the affected cell types. This is already utilized to study pathomechanisms in a variety of tissues and cell types [9, 16–18]. The so-called channelopathies which are based on mutations in ion channels are the cause of developmental disorders and are the subject of various studies [19, 20]. To elucidate the role of ion channels in cell differentiation, maturation or their role in pathomechanisms a well-founded knowledge of ion channel distribution in pluripotent cells, representing one of the earliest stages of development, is indispensable. In that respect, we compared the expression levels of several ion channels in human keratinocytes with their reprogrammed progeny, hiPSCs. Keratinocytes derived from plucked human hair [21, 22] represent one of the most promising cell sources for the generation of the investigated hiPSCs . We have elucidated ion channel regulation for various channel families and their subtypes. Additionally, we compared the expression levels of ion channel families and subtypes, found to be regulated during reprogramming, with iPSC-differentiated progeny, namely, neurons and cardiomyocytes. These comparisons could be a start point to evaluate the contribution and function of different ion channels, for example, for self-renewal and differentiation processes in stem cells.
2. Material and Methods
2.1. Ethical Statement and Donor Information
After informed consent was given by the donors (ethical agreement by the University of Ulm, number 88/12) hair with intact hair roots was gathered by plucking from the scalp after desinfection. We used hair from healthy volunteers (age between 24 to 45 and both male and female gender).
2.2. hiPSC Generation and Cell Culture
Keratinocytes were obtained from plucked human scalp hair as already described . Keratinocytes were propagated in EpiLife medium with HKGS supplement (both Invitrogen, Carlsbad, CA, USA). hiPSCs were generated from keratinocytes by lentiviral transduction of four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) as described earlier . After the reprogramming on rat embryonic fibroblasts they were maintained feeder-free on Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) coated dishes in mTeSR1 medium (Stemcell Technologies, Vancouver, CA, USA).
2.3. Gene Expression Microarrays
Gene expression microarrays were performed for 6 keratinocyte samples and 9 hiPSC samples with the Agilent Whole Human Genome Microarray Kit (4x44k microarray kit G4112F, Agilent Technologies, Santa Clara, CA, USA). 500 ng of total RNA was used to produce Cy3-CTP-labeled cRNA with the Agilent Low RNA Input Liner Amplification Kit. The cRNA was purified and 1,65 μg per array was hybridized for 17 h at 65°C and 10 r.p.m. Afterwards, the arrays were washed with Agilent Gene Expression Wash Buffers one and two and finally with acetonitrile for 1 min. The slides were scanned using Scan Control 7.0 software with a resolution of 5 μm. Scan data was extracted with the Feature Extraction 9.1 software. Expression levels were background adjusted and quantile normalized with the GeneSpring GX 12 software. Differential expression between keratinocytes and hiPSCs was analyzed using student’s t-test. A fold change > 2 and a value < 0.05 was considered significant and highlighted bold (upregulation) or italic (downregulation) in the results table. For comparisons of iPSCs with iPSC-derived neurons published data from GSE34879 (GSM856936, GSM856937, GSM856915, GSM856916) and for cardiomyocytes GSE17579 (GSM438022, GSM438026, GSM438034, GSM438021, GSM438032, GSM438036) were used (both from NCBI Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/geo/). Fold change is shown if >2.
3.1. Differential Expression of Ion Channels in hiPSCs Compared to Keratinocytes
We first compared the expression of various ion channel families from keratinocytes to hiPSCs and from hiPSCs to cardiomyocytes and neurons, respectively. From the 387 probes (Table 1) binding in ion channel genes from parental keratinocytes to hiPSCs, 101 (26%) showed a significant increase in expression (fold change > 2, ; labeled in bold) while 23 (6%) showed a significant decrease (fold change > −2, ; labeled in italic). In a second step differentially regulated ion channels from hiPSCs to hiPSC-derived neurons were investigated and we found 29 ion channel transcripts to be upregulated (fold change > 2; labeled in bold) while 6 showed a significant decrease (fold change > −2; labeled in italic). For cardiomyocytes, mRNA levels of only 7 ion channel members were upregulated (fold change > 2; labeled in bold) and 10 mRNA levels showed a significant decrease (fold change > −2; labeled in italic).