Using human induced pluripotent stem cells to investigate neurodevelopmental effects of human cytomegalovirus

Human cytomegalovirus (HCMV) is one of the leading prenatal causes of mental retardation and congenital deformities, world-wide. Its pathogenesis has generally been investigated using animal models. Human studies in vitro have been limited to neurospheres prepared using forebrain tissues from fetal abortuses. This approach is limited and does not permit analysis of individual specific cells. We generated iPS cells from adult human fibroblasts. iPS cells were differentiated into neurospheres, that were expanded as monolayer culture of neuroprogenitors (NPs). Furthermore, neurospheres were differentiated into neurons that could be stained for Tuj1, tyrosine hydroxylase and NR4A2. Functional competency was confirmed by live imaging of intracellular calcium. NPs and neurons were infected with HCMV (MOI = 3). Cell viability was assessed by FACS analysis. Cytopathic effects of HCMV were observed on the 10th day post infection in neuroprogenitor cells. Earlier, the adherence of these cells to the matrix was reduced. Neurons were much more refractory. Reduced cell density and shortening of neuritic processes was only observed at day 15 after infection. We are presently examining the intracellular effects of HCMV. Human iPS cells can efficiently generate neurospheres, which can be expanded as almost pure cultures of neuroprogenitors or differentiated into neurons. iPS cells-derived NP and neurons offer powerful cellular models to investigate the effect of neurotropic viral agents on neurodevelopment

High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells

Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs) by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM), and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP) 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications. © 2014 Ao et al

Dynamic gene expressions during hepatic differentiation from piPSCs.

<p>Q-PCR analysis of pluripotency marker genes (Oct4, Nanog, and Sox2), definitive endoderm markers (FoxA 2, GATA4 and Sox17), hepatic progenitor markers (AFP, TTR and HNF 4α), hepatocyte markers (ALB, HNF 1α, and CK18), metabolizing phase I enzymes (CYP3A29, CYP2C34, CYP1A1), phase II enzymes (GST A1, GST A2, GST A4) and phase III transporters (MRP1, GLUT2, and P-gp3) at different time points of differentiation with the two hepatic differentiation methods. The ratio of ΔΔCT was normalized to the internal control GAPDH, and fold change results were obtained by normalization to undifferentiated piPSCs on T0. Error bars represent SEM of three independent experiments. ^*<i>P</i><0.05, ^**<i>P</i><0.01. <i>t</i>-test.</p

ALB and AFP protein expressions in hepatocyte-like cells.

<p>(A) The expressions of ALB and AFP in undifferentiated piPSCs, T18 hepatocyte-like cells generated from Method I & II and pig liver tissue, were detected by immunostaining. Scale bars, 100 µm. The ratios of ALB and AFP positive cells were quantified from approximately 300 cells of each sample. <i>P</i><0.05. (B) The expressions of ALB and AFP on above samples were next examined by using western blot assay. The relative expression ratio was normalized to the internal control GAPDH. Error bars represent SEM of three independent experiments. P value was calculated using Student's <i>t</i>-test.</p

Differentiation of hepatocyte-like cells from piPSCs.

<p>(A) A protocol outline describing Method I of hepatocyte differentiation from piPSCs. (B) Morphological changes during piPSCs differentiation to hepatocyte-like cells using Method I. (C) A protocol outline describing Method II of hepatocyte differentiation from piPSCs. (D) Morphological changes during piPSCs differentiation to hepatocyte-like cells using Method II.</p

Generation of piPSCs from PEFs.

<p>(A) From left to right, morphology of PEFs, an induced piPSCs colony, and an iPSCs colony post AP staining. Scale bars, 200 µm. (B) Typical iPSC colonies from mouse, pig and human before passaging. Scale bars, 200 µm. (C) Expression of pluripotency markers of Oct4, Nanog, Sox2, SSEA1, SSEA4, TRA 1-60, and TRA 1-81 by immunostaining. Scale bars, 200 µm. (D) Q-PCR analysis of pluripotency marker genes Oct4, Nanog and Sox2 in PEFs (red) and piPSCs (blue). The ratio of ΔΔCT was normalized to the internal control GAPDH, error bars represent SEM of three independent experiments. (E) Histological analysis of teratoma derived from piPSCs. Ectoderm (a): pigment epithelium; Mesoderm (b): muscle; Endoderm (c): ciliated columnar epithelium. (F) Karyotyping analysis show the normal karyotype of piPSCs. Error bars show SEM of three independent experiments. P value was calculated using Student's <i>t</i>-test.</p

Functional analysis of the hepatocyte-like cells derived from piPSCs.

<p>(A) Analyzing LDL uptake on undifferentiated piPSCs, T18 hepatocyte-like cells generated from Method I & II and primary pig hepatocytes. Red fluorescence indicates the cytosolic LDL. Scale bars, 100 µm. (B) PAS staining assay to examine glycogen storage on undifferentiated piPSCs, T18 hepatocyte-like cells generated from Method I & II and pig liver tissue. Scale bars, 200 µm; Scale bars of high magnification images, 25 µm. (C) Urea concentration on T14, T16 and T18 piPSC-derived hepatocyte-like cells using two different differentiation methods. Undifferentiated piPSCs serve as a negative control. (D) Activities of CYP3A and CYP2C on the T18 piPSC-derived hepatocyte-like cells using two differentiation methods. Error bars represent SEM of three independent experiments. P value was calculated using Student's <i>t</i>-test. Undifferentiated piPSCs serve as a negative control.</p

Upregulation of keratocyte-specific gene expression in pellet cultures.

<p>Expression of six genes, previously identified as up-regulated during keratocyte differentiation, was determined after 2 weeks in pellet cultures derived from either MEM+FBS or N2 monolayer cultures as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. Gene expression is calculated relative to the NGFR+ derived hES cells. Error bars represent S.D. of triplicates. All genes were significantly (p<0.05) upregulated in pellets compared to NGFR+ cells except for CHST6. Asterisks show cases in which pellet culture induced a significant (p<0.05) increase in gene expression compared to the monolayers cultures.</p

Secretion of corneal keratan sulfate proteoglycans by pellet cultures.

<p>Proteoglycans were isolated from culture medium before (Lanes 1,2,5,6) or after (3,4,7,8) three-week incubation with hES pellets. The proteoglycan fractions were biotin- labeled and immune-precipitated with antibodies against keratocan (anti-Kera) (lanes 1–4) or keratan sulfate glycosaminoglycan (anti-KS) (lanes 5–8). Half of each sample was digested with endo-ß-galactosidase (as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>) to hydrolyze keratan sulfate, and samples were separated by SDS-PAGE, transferred to PVDF membranes and biotinylated proteins detected with avidin-labeled infrared dye as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. Presence of biotinylated proteins migrating as a broad, heterogeneous >100 band typical of keratan sulfate proteoglycan (KSPG - bracket on the left) was present in lanes 3 and 7. Sensitivity of this material to digestion with keratan sulfate-specific glycosidase (lanes 4 and 8) demonstrates presence of keratocan-linked keratan sulfate, a unique keratocyte biosynthetic product.</p

Co-culture with PA6 cells induces upregulation of neural crest gene expression in hES cells.

<p>hES cells were co-cultured with PA6 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a>. RNA was isolated at post-induction days (PID) 2, 4, 6, and 8. Expression of characteristic neural crest (NC) marker genes was determined by qPCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#s2" target="_blank">Methods</a> using human-specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056831#pone-0056831-t002" target="_blank">Table 2</a>). Expression levels are calculated relative to untreated hES cells (hES = 1). Error bars show the standard deviation (S.D.) of triplicate analyses.</p