Equivalence of Conventionally-Derived and Parthenote-Derived Human Embryonic Stem Cells

As human embryonic stem cell (hESC) lines can be derived via multiple means, it is important to determine particular characteristics of individual lines that may dictate the applications to which they are best suited. The objective of this work was to determine points of equivalence and differences between conventionally-derived hESC and parthenote-derived hESC lines (phESC) in the undifferentiated state and during neural differentiation.hESC and phESC were exposed to the same expansion conditions and subsequent neural and retinal pigmented epithelium (RPE) differentiation protocols. Growth rates and gross morphology were recorded during expansion. RTPCR for developmentally relevant genes and global DNA methylation profiling were used to compare gene expression and epigenetic characteristics. Parthenote lines proliferated more slowly than conventional hESC lines and yielded lower quantities of less mature differentiated cells in a neural progenitor cell (NPC) differentiation protocol. However, the cell lines performed similarly in a RPE differentiation protocol. The DNA methylation analysis showed similar general profiles, but the two cell types differed in methylation of imprinted genes. There were no major differences in gene expression between the lines before differentiation, but when differentiated into NPCs, the two cell types differed in expression of extracellular matrix (ECM) genes.These data show that hESC and phESC are similar in the undifferentiated state, and both cell types are capable of differentiation along neural lineages. The differences between the cell types, in proliferation and extent of differentiation, may be linked, in part, to the observed differences in ECM synthesis and methylation of imprinted genes

Equivalence of Conventionally-Derived and Parthenote-Derived Human Embryonic Stem Cells

BackgroundAs human embryonic stem cell (hESC) lines can be derived via multiple means, it is important to determine particular characteristics of individual lines that may dictate the applications to which they are best suited. The objective of this work was to determine points of equivalence and differences between conventionally-derived hESC and parthenote-derived hESC lines (phESC) in the undifferentiated state and during neural differentiation.Methodology/Principal FindingshESC and phESC were exposed to the same expansion conditions and subsequent neural and retinal pigmented epithelium (RPE) differentiation protocols. Growth rates and gross morphology were recorded during expansion. RTPCR for developmentally relevant genes and global DNA methylation profiling were used to compare gene expression and epigenetic characteristics. Parthenote lines proliferated more slowly than conventional hESC lines and yielded lower quantities of less mature differentiated cells in a neural progenitor cell (NPC) differentiation protocol. However, the cell lines performed similarly in a RPE differentiation protocol. The DNA methylation analysis showed similar general profiles, but the two cell types differed in methylation of imprinted genes. There were no major differences in gene expression between the lines before differentiation, but when differentiated into NPCs, the two cell types differed in expression of extracellular matrix (ECM) genes.Conclusions/SignificanceThese data show that hESC and phESC are similar in the undifferentiated state, and both cell types are capable of differentiation along neural lineages. The differences between the cell types, in proliferation and extent of differentiation, may be linked, in part, to the observed differences in ECM synthesis and methylation of imprinted genes

phESC produce functional RPE cells after differentiation.

<p>The LLC6P line produces pigmented cells in culture (a) and characteristic “dome-like” structures (b). Cells express RPE markers Occludin (c), CRALBP and mitf (d). Functionality of LLC6P-derived RPE is demonstrated by pHrhodo pH-sensitive fluorescent particle phagocytosis (e and d). Five minutes after administration of pHrhodo (e), RPE exhibit minimal vesicular pHrhodo content. Two hours and five minutes after administration (f), RPE exhibit marked increase in vesicular pHrhodo content. Scale bars represent 100 µm (a, e, f), 500 µm (b) and 5 µm (c, d).</p

Undifferentiated colonies differed with respect to imprinted genes.

<p>PCR arrays did not reveal differences in the undifferentiated state (a). Parthenote-derived lines LLC6P and LLC8P are plotted on the y-axis and conventionally-derived lines H7 and CSC14 on the x-axis. Methylation microarray revealed differences between parthenote-derived lines and conventionally-derived lines, most notably in imprinted genes. Cluster analysis (b) indicated that phESC and hESC were closely related to each other and more methylated than tissue and primary cell lines. phESC and hESC are distinguishable when complete methylation data are included (c). phESC and hESC diverge further when methylation data include only imprinted genes (d). Methylation data, normalized with tissue lines as the unmethylated baseline (c and d). In c and d, correlation is plotted on the y-axis and level of discordance between 7 phESC lines is plotted on the x axis (e.g. 4 means 3 phESC lines were allowed discordance). Red represents phESC lines, green represents hESC lines, blue represents primary cell lines and dashed purple represents tissue lines.</p

phESC display altered ECM profile and do not readily form spheres in non-adherent culture.

<p>During the sphere-forming stage of the NPC protocol, hESC gave rise to clusters that readily formed spheres with minimal dissociation (a). phESC at the same stage consistently dissociated (b). PCR arrays revealed differences in extracellular matrix and adhesion molecules in NPC (c). Parthenote-derived lines LLC6P and LLC8P are plotted on the y-axis and conventionally-derived lines H7 and CSC14 on the x-axis. Details of differentially expressed genes are presented numerically (d). Western blot analysis confirmed differential expression of NCAM1 (e). Scale bars represent 20 µm (a–b).</p

hESC and phESC differ morphologically.

<p>Conventionally-derived (a–f) and parthenote-derived (g–l) hESCs have distinct morphology and growth patterns in the undifferentiated state. High magnification and low magnification images have been provided for each colony to illustrate the slower growth of parthenote-derived colonies, irregular colony shape and lack of stromal cells (long, thin cells between colonies indicated by arrows) in the phESC cultures. Day 1 after plating (a, d, g and j). Day 2 after plating (b, e, h and k). Day 4 after plating (c, f, i and l). Scale bars represent 20 µm (a–c and g–i) and 10 µm (d–f and j–l).</p

Immunocytochemical profile following NPC differentiation.

<p>Parthenote-derived lines displayed impaired neural maturation, but expressed immature markers normally. CSC14 (a–d), H7 (e–h), LLC6P (i–l) and LLC8P (m–p). Map2 (a, e, i and m), Olig1 (b, f, j and n), Nestin (c, g, k and o) and Pax6 (d, h, l and p). Scale bars represent 50 µm (a, b, e and f) and 10 µm (c, d, and g–p).</p

Exposure of hESC and phESC to a neural differentiation protocol yields quantitatively distinct cell populations.

<p>Conventionally-derived (a and b) and parthenote-derived (c and d) hESC have distinct growth patterns after the NPC differentiation protocol. Histograms plotting cellular yield (in millions of cells) after NPC differentiation (e) reveal that parthenote-derived lines yield fewer cells after differentiation than conventionally-derived lines. Scale bars represent 10 µm. Blue bars indicate differentiation 1, red bars indicate differentiation 2 and yellow bars indicate differentiation 3, representing the three successive differentiations.</p