mES, 4-mAdiPS and 2-mAdiPS cells exhibit similar morphology, gene expression and pluripotency. (A–L) Similar morphology, alkaline phophatase histochemical staining, Oct4 and SSEA-1 immunoreactivity for R1 mES, 4-mAdiPS and 2-mAdiPS cells (scale bars, 50 µm). (M) RT–PCR analysis shows similar Nanog, Oct4 and Sox2 expression in mES, 4-mAdiPS and 2-mAdiPS cells. (N) Western-blot expression of Oct4 in 4-mAdiPS cells at the expected 40 kDa size. (O) Teratoma derived from 4-mAdiPS cells injected into SCID mouse. (P and Q) Differentiation of histologically distinct tissue types in teratomas derived from 4-mAdiPS cells injected into SCID mice, assessed by H&E staining, demonstrates glandular and smooth muscle differentiation (scale bars, 200 µm). (R–T) Positive immunostaining for AFP, SMA and NF in teratomas derived from 4-mAdiPS cells injected into SCID mice. (U–W) 2-mAdiPS cells differentiated in vitro for 7 days exhibit similar immunoreactivity for AFP, SMA and NF (scale bars, 100 µm). mES, mouse embryonic stem cells; Alk. Phos., alkaline phosphatase; mAmnio, mouse amniocytes; MEFs, mouse embryonic fibroblasts; AFP, alpha-fetoprotein; SMA, smooth muscle actin; NF, neurofilament.

(A) Dendrogram and (B) heatmaps showing relationship between two independent samples of each of hES cells, hAdiPS cells and human amniocytes. The expression values within heatmaps represent the fold difference of expression from the ES cell lines calculated by the logarithm (base 2) of the ratio between the expression level for a gene between the average expression of the ES cell lines for that particular gene. Heatmaps were prepared using unsupervised hierarchical clustering.

Mitotically inactivated amniocytes function as feeder layers for mES, hES and hAdiPS cells. (A and B) Mitomycin C-treated mouse amniocytes function as feeder layer for R1 mES cells. After 10 passages (30 days) in culture, mES cell colonies demonstrate AP activity and BrdU labeling. (C and D) Oct4-GFP reporter fluorescence and Oct4 immunostaining of mES colonies cultured for 30 days on amniocyte feeders. (E and F) NF and SMA immunostaining after 7 and 14 days of in vitro differentiation, respectively. (G) RT–PCR analysis of Oct4, Nanog and Sox2 expression in early (E, <3) and late (L, >10) passage mES cells maintained on MEFs or mouse amniocytes. Controls (*) are feeders without mES cells. (H) Germline competent chimera from blastocyst injection of mES cells maintained for 50 days (15 passages) on mouse amniocyte feeder layer. (I–K) OCT4, SSEA-4 and TRA-1-81H1 immunoreactivity in hES cells cultured for 21 days (six passages) on γ-irradiated human amniocytes. (L–N) AFP, SMA and NF immunostaining after EB differentiation of H1 hES cells cultured on γ-irradiated human amniocytes. (O–T) γ-irradiated human amniocyte feeder layers support growth and self-renewal of hAdiPS cells derived from the same amniocytes. After 21 days (five passages) in culture, hAdiPS cell colonies demonstrate AP activity and OCT4, SSEA-4, TRA-1-60 and TRA-1-81 expression by immunocytochemistry. mES and hES cells, mouse and human embryonic stem cells; AP or Alk. Phos., alkaline phosphatase; BrdU, bromodeoxyuridine; Amnios, mouse amniocytes; MEFs, mouse embryonic fibroblasts; EP, early passage; LP, late passage; AFP, alpha-fetoprotein; SMA, smooth muscle actin; NF, neurofilament. Scale bars: 100 μm.

Reprogramming protocol and infection efficiency of mouse and human amniocytes. (A) Mouse or human amniocytes were plated (day −3) in the absence of feeder layers and infected (day 0) with four (Oct4, Sox2, Klf4, c-Myc) or two (Klf4, c-Myc) retroviral vectors, with colonies emerging by 5 days (four factors, mouse and human) or 7 days (two factors, mouse) post-infection. Colonies were picked on day 9 via live immunostaining and transferred to feeder layers. (B) Phase image of cultured mouse amniocytes 5 days after plating. (C) The efficiency of retroviral infection of cultured amniocytes was >90% as reflected by retroviral GFP reporter expression 2 days post-infection. (D) A single primitive hAdiPS cell colony on day 5 post-infection (scale bars, 200 µm).

hES and hAdiPS cells exhibit similar morphology and gene expression. (A–J) Similar morphology and OCT4, SSEA-4, TRA-1-60 and TRA-1-81 immunoreactivity for H1 hES cells and hAdiPS cells (scale bars, 100 µm). (K) Transcripts for OCT4, SOX2, GDF3, DNMT3B and REX1 are expressed at comparable levels in hES and hAdiPS cells. (L) Microarray analysis of hAdiPS cell gene expression compared with H1 hES cells demonstrating 0.99 correlation as opposed to 0.82 for human amniocytes relative to H1 hES cells. (M) Bisulfite-treated PCR-amplified sequence of the promoter regions of OCT4 and NANOG. Open and closed circles indicate unmethylated and methylated CpGs. hES, human embryonic stem cells; hAmnio, human amniocytes; MEFs, mouse embryonic fibroblasts.

hAdiPS cells demonstrate pluripotency. (A–C) hAdiPS cells differentiated in culture for 14 days exhibit subpopulations of cells immunoreactive for AFP, SMA and NF. (D–F) Similar immunoreactivity is observed when cryosections of EBs from hAdiPS cells are analyzed (scale bars, 100 µm). (G–I) Analysis of teratomas derived from hAdiPS cells injected in SCID mice shows differentiation of histologically distinct tissue types such as glandular tissue (endoderm), muscle tissue (mesoderm) and neuroepithelial tissue (ectoderm), respectively (scale bars, 100 µm). (J–L) Positive immunostaining for AFP, SMA and NF in teratomas derived from hAdiPS cells injected into SCID mice (scale bars, 50 µm). EBs, embryoid bodies; AFP, alpha-fetoprotein; SMA, smooth muscle actin; NF, neurofilament; SCID, severe combined immunodeficient mice.

Patient-specific stem-cell therapy using human amniocytes in a xenoculture-free, autologous system. In this model, discarded amniocytes are obtained as a byproduct of routine antenatal screening and can be banked. At an appropriate time, they are cultured and reprogrammed in vitro to become patient-specific amniocyte-derived induced pluripotent stem (AdiPS) cells. AdiPS cells may then be maintained and expanded on amniocyte feeder cells from the same patient and subsequently banked for later use in cell-based directed differentiation or other protocols to generate new tissues.