The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that co-differentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). In order to better characterize the iPGCs we performed Real time PCR, microarray and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure.

In two validation studies, TELiS accurately detected in vivo activation of NF-kappaB and the Type I interferon system by HIV-1 infection and pharmacologic activation of the glucocorticoid receptor in peripheral blood mononuclear cells. The population-based statistical inference underlying TELiS out-performed conventional statistical tests in analytic sensitivity, with parametric studies demonstrating accurate identification of transcription factor activity from as few as 20 differentially expressed genes. TELiS thus provides a simple, rapid and sensitive tool for identifying transcription control pathways mediating observed gene expression dynamics.

IntroductionChemokine receptor CCR5 is an attractive therapeutic target for inhibiting HIV-1, as it serves as a HIV-1 coreceptor and is essential for CCR5 tropic HIV-1 infection. [1][2][3][4] Blocking CCR5 expression should prevent HIV-1 infection at the initial stage of the viral life cycle. Individuals with a ⌬32/⌬32 homozygous mutation in the CCR5 gene do not express CCR5, are highly protected from HIV-1, and are apparently normal. [5][6][7] Recently, an HIV ϩ acute myelogenous leukemia patient was treated for leukemia and HIV infection by bone marrow transplantation using donated CCR5 ⌬32/⌬32 marrow. After the transplantation, nearly 100% of the patient's blood cells were replaced with donor cells. HIV DNA and RNA were undetectable at 20 months, even after the discontinuation of highly active antiretroviral therapy. 8 This evidence supports that long-term and stable reduction of CCR5 is a promising strategy for treating HIV-infected patients. The major limitation of this strategy is the difficulty of identifying human leukocyte antigen-matched CCR5 ⌬32/⌬32 homozygous donors as the mutation exists in approximately 1% of white populations and is rare in other ethnic populations. 9 Small interfering RNAs (siRNAs) induce sequence-specific degradation of mRNAs by RNA interference. 10 Many forms of siRNA have been used to inhibit HIV coreceptors and HIV-1 gene expression in in vitro and in vivo experimental settings. [11][12][13][14][15][16][17][18] To stably inhibit HIV replication, we and others developed lentiviral vectors that are capable of stably delivering short hairpin RNA (shRNA) in mammalian cells. [19][20][21][22][23][24][25] We demonstrated that expression of CCR5-specific shRNA in human primary T lymphocytes results in efficient CCR5-knockdown and protection of cells from HIV-1 infection in vitro. 22 However, we and others recognized that a high level of sustained shRNA expression may be toxic to cells because of competition with endogenous micro-RNA biogenesis, induction of interferon responses, and/or off-targeting effects. 23,[26][27][28][29][30][31][32][33] To stably reduce CCR5 expression without cytotoxicity, we identified a highly efficient shRNA (shRNA 1005) directed to human CCR5 mRNA using the enzymatic production of RNAi libraries (EPRIL) screening technique. 21,34 We expressed shRNA 1005 using the transcriptionally weak H1 promoter to stably reduce CCR5 expression without inducing cytotoxicity in human primary peripheral blood lymphocytes in vitro. 21,34 To test stable CCR5 reduction in vivo, we used a nonhuman primate hematopoietic stem cell transplantation model in which we were able to demonstrate stable reduction of CCR5 expression in peripheral blood lymphocytes in shRNA-transduced CD34 ϩ cell-transplanted rhesus macaques. 21 Because of a single nucleotide mismatch in the shRNA 1005 target sequence between human and rhesus macaque CCR5 mRNA, we mutated the human CCR5 shRNA 1005 so that it would be 100% homologous to the corresponding rhesus macaque CCR5 mRNA target sequence. This rhes...

SummaryJoint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166low/negCD146low/negCD73+CD44lowBMPR1B+) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5–6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166low/negBMPR1B+ putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering.