Dynamic Changes of Pectin Epitopes in Cell Walls during the Development of the Procambium–Cambium Continuum in Poplar

The change of pectin epitopes during procambium–cambium continuum development was investigated by immunolocalization in poplar. The monoclonal antibody JIM5 labels homogalacturonan (HGA) with a low degree of esterification, and the monoclonal antibody JIM7 labels HGA with a high degree of methyl-esterification. Arabinan, rather than galactan, and HGA with low degree of esterification were located in the cell walls of procambial, while HGA with a low degree of esterification was located in the tangential walls, and galactan was located in both the tangential and radial walls of procambial, yet nearly no arabinan was located in the tangential walls of the cambial cells. The changes in pectin distribution took place when periclinal divisions appeared within a procambial trace. The distribution difference of pectin epitopes was also present in procambium–cambium derivatives. The arabinan existed in all cell walls of primary xylem, but was absent from the tangential walls of secondary xylem cells. The galactan existed only in mature primary phloem. Furthermore, 19 pectin methylesterases (PMEs) genes were identified by RNA sequencing, six genes presented highly differentially and were supposed to be involved in the cell wall esterification process. The results provide direct evidence of the dynamic changes of pectin epitopes during the development of the procambium–cambium continuum in poplar

Optimized Approaches for Generation of Integration-free iPSCs from Human Urine-Derived Cells with Small Molecules and Autologous Feeder

Generation of induced pluripotent stem cells (iPSCs) from human urine-derived cells (hUCs) provides a convenient and non-invasive way to obtain patient-specific iPSCs. However, many isolated hUCs exhibit very poor proliferation and are difficult to reprogram. In this study, we optimized reprogramming approaches for hUCs with very poor proliferation. We report here that a compound cocktail containing cyclic pifithrin-a (a P53 inhibitor), A-83-01, CHIR99021, thiazovivin, NaB, and PD0325901 significantly improves the reprogramming efficiency (170-fold more) for hUCs. In addition, we showed that replacement of Matrigel with autologous hUC feeders can overcome the reprogramming failure due to the massive cell death that occurs during delivery of reprogramming factors. In summary, we describe improved approaches to enable iPSC generation from hUCs that were otherwise difficult to reprogram, a valuable asset for banking patient-specific iPSCs

Characterization of a typical non-intergrated iPS cell line generated from UC-012.

<p>A. Non-integrating analysis of eipsomal DNA in the iPS cells. −: negative control. +: positive control, UCs transfected with indicated episomal vectors. UC-012: urine-derived cells 12. UiPSC-012 C2P19 and UiPSC-012 C6P22: iPS cell lines, C: colony number, P: passage. B. G-band analysis of the iPS cells derived from UC-012 shows normal karyotype. C. qPCR for endogenous human ES cell specific transcription factors of UC-012 and two derivative iPS cell lines. UC-015 and UC-041 and derivative iPS cell lines were also shown. Values were referred to 10⁶ copies of <i>ACTIN</i>. Human ES cell line H1 was used as control. n = 3. <i>P</i> value is referred to UCs. ** indicates <i>P</i><0.01. D. Expression of human ES markers OCT4 and SSEA4 of UiPSC-012 C2P19 by flow cytometry. E. Immunofluorescence for human ES markers of UiPSC-012 C2P18. F. Methylation status of <i>OCT4</i> and <i>NANOG</i> promoters in UC-012 and UiPSC-012 C2P19. G. Top: EB formed from UiPSC-012 C2P18. Bottom: qPCR analysis of human ES cell markers and markers for the three germ layers relative to iPSCs. n = 3. * indicates <i>P</i><0.05. H. HE-staining of the teratomas from UiPSC-012 C2P17.</p

List of other UC lines.

*<p>M: male F:female.</p>**<p>4/8:4 out of 8.</p

List of UiPSCs generated from UCs.

<p>List of UiPSCs generated from UCs.</p