Development of a focused microarray to assess human embryonic stem cell differentiation.
Human embryonic stem cells (hESC) must be differentiated before clinical use. In addition, the extent of contamination of undifferentiated cells and the efficiency of differentiation must also be assessed prior to clinical application. In this manuscript, we describe the development of a focused microarray that may be used to discriminate between hESC and their differentiated progeny. This array contains 755 genes including embryonic stem cell markers as well as markers of differentiation into neural, mesodermal, and endodermal phenotypes. In addition, we have included candidate genes belonging to families of cytokines, chemokines, receptors, signaling pathways, and homeodomain proteins that are likely to be important in the process of differentiation. Testing and validation of the focused array was performed using RNA from hESC, human embryoid body (hEB) outgrowths, and a human embryonal carcinoma (hEC) cell line. We have compared gene expression with negative background, GAPDH, beta-actin positive controls, and human universal RNA (hURNA), showing that such an array can rapidly distinguish between undifferentiated and differentiated hESC-derived cell populations. We expect that the described array will be extremely useful in evaluating the extent of differentiation and the state of the hESC-derived population utilized for therapeutic purposes.
Yang
Amy X
AX
Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Mejido
Josef
J
Luo
Yongquan
Y
Zeng
Xianmin
X
Schwartz
Catherine
C
Wu
Tianxia
T
Thies
R Scott
RS
Bhattacharya
Bhaskar
B
Han
Jing
J
Freed
Bill
B
Rao
Mahendra
M
Puri
Raj K
RK
D002454:Cell Differentiation; D002460:Cell Line; D004622:Embryo, Mammalian; D004798:Enzymes / Q000235:genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011506:Proteins / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology
D004798:Enzymes; D011506:Proteins; D012313:RNA