Figure 1.
Expression and synthesis of laminins by JAR cells.
A) JAR choriocarcinoma cells transcribe lm α1, lm α3, lm α5, lm β1, lm β2, lm β, lm γ2, and lm γ1 chains as shown by RT-PCR. As a reference sample, we used optimized, in-house prepared positive control mix, which contains cDNA from undifferentiated hPSCs, spontaneously differentiated hPSCs as well as from isolated human pancreatic islets. B) JAR choriocarcinoma cells were metabolically labeled with 35S and the cell culture supernatant and matrix deposited by JAR cells were immunoprecipitated using antibodies, which specifically recognize the human lm α1 (400 kDa) and lm α5 (350-380 kDa) chains. The cells showed abundant lm-511 and -111 synthesis.
Figure 2.
JAR matrix maintains hPSCs undifferentiated and pluripotent.
A) Two hPSC lines, FES29 and HEL11.4, were cultured on JAR matrix or Matrigel for 15 passages and characterized in the beginning (p0), at p5 and at p15. Expression of genes typical for undifferentiated hPSCs (OCT4, SOX2, NANOG) and early differentiation markers (SOX1, SOX17 and BRACHYURY) were analyzed by qPCR. Each data point represents the pooled sample from five replicate plates per cell line.
B) The hPSC lines were analyzed by flow cytometry in the beginning (p0), at p5 and at p15. The levels of SSEA3, TRA1-60 and H1 type antigen were high throughout the culture period, while the level of SSEA1 remained low, suggesting that pluripotent hPSCs were maintained on JAR matrix equally well as on Matrigel. Each time point represents the pooled sample from five replicate plates per cell line.
C) Morphology of the hiPSC line HEL11.4 on JAR matrix and on Matrigel. Phase contrast micrographs and immunofluorescent staining of the hiPSC line HEL11.4 with antibodies recognizing OCT4, Tra1-60 and NANOG, after culture for 15 passages on JAR matrix. Scale bars: 500 µm for the upper phase contrast image of HEL11.4. on JAR matrix, others 100 µm.
D) hPSCs formed typical embryoid bodies after 15-passage culture on JAR matrix. Presence of endodermal (SOX17), ectodermal (BETA(III)TUBULIN/TUJ1) and mesodermal (VIMENTIN) derivatives was detected by immunofluorescence in hiPSC line HEL 11.4. Scale bar 100 µm.
E) Mature derivatives of all germ layers; neuroectoderm, endoderm and mesoderm in a teratoma obtained from the hiPSC line HEL11.4 after culture on JAR matrix for 15 passages. Objective magnification 40X.
Figure 3.
JAR matrix allows hPSC differentiation to neuronal lineages.
A) The qPCR results show that the expression level of OCT4 declined during directed neuronal differentiation on JAR matrix and Matrigel while the expression levels of SOX1 and PAX6 increased. Expression level is relative to an in-house prepared positive control mix, which contains cDNA from undifferentiated and differentiated hPSCs and human pancreatic islets. Data represent the mean (±SD) of results from two independent experiments with the hPSC line H9.
B) Phase contrast images of the H9 cells cultured on JAR matrix prior to onset of differentiation (upper panel). Scale bars 500 µm (left) and 100 µm, (right). After a 7-day differentiation, the majority of the hPSCs had differentiated to PAX6 and/or BETA(III)TUBULIN positive cells. The data represents the hPSC line H9, cultured for 5 passages on JAR matrix before the differentiation.
Figure 4.
JAR matrix supports hPSC differentiation to hepatocyte like cells.
A) qRT-PCR analysis of the expression levels of hepatocyte differentiation markers, AFP and ALBUMIN, during differentiation. Data represent the mean (±SEM) of three independent experiments on H9 and HEL11.4 cells.
B) The morphology (upper panel) and immunocytochemistry for ALBUMIN and AFP (lower panel) of the HEL11.4 cells after differentiation. Scale bars 100 µm.
C) Quantitative flow cytometry analysis of the cells after differentiation to hepatocyte like cells on JAR matrix or Matrigel. The results are representative for three independent experiments.
Figure 5.
JAR matrix supports adhesion of the early hiPSC colonies.
A) Formation of the hiPSC colonies on JAR matrix (left panel) and Matrigel (right panel) at day 5, 7, 9, 11 and 13 after retroviral transduction. Objective magnification 4X.
B) The hiPSC colonies from three independent retroviral inductions on JAR matrix and Matrigel were stained for TRA1-60 on day 14 and imaged by the Cell-IQ imaging system. There was no difference in the number of colonies. N=3.
C) Twelve clones from three independent inductions were picked and plated either on JAR matrix or Matrigel. The clones adhered significantly better on JAR matrix (left). There was no significant difference on early differentiation of the clones on JAR matrix and Matrigel (right). N=3.
Figure 6.
Characterization of new hiPSC lines generated on JAR-matrix.
A) Human foreskin fibroblasts were induced to pluripotency by the Sendai virus reprogramming kit. Both hiPSC lines showed endogenous expression of OCT4, SOX2 and NANOG but were free from sendai virus replicon when analyzed at passage 12.
B) The hiPSC lines generated on JAR matrix were stained for presence of TRA1-60 and NANOG. Both of the hiPSC lines were positive for the pluripotency markers. Scale bar 100 µm.
C) Undifferentiated hiPSC2 cells were transplanted into testes of nude mouse. The hiPSC 2 cells had formed well-matured teratoma, which contained endodermal, mesodermal and neuroectodermal derivatives.