Parthenogenetic activation of human oocytes may be one way to produce histocompatible cells for cell-based therapy. We report the successful derivation of six pluripotent human embryonic stem cell (hESC) lines from blastocysts of parthenogenetic origin. The parthenogenetic human embryonic stem cells (phESC) demonstrate typical hESC morphology, express appropriate markers, and possess high levels of alkaline phosphatase and telomerase activity. The phESC lines have a normal 46, XX karyotype, except one cell line, and have been cultured from between 21 to 35 passages. The phESC lines form embryoid bodies in suspension culture and teratomas after injection to immunodeficient animals and give differentiated derivatives of all three embryonic germ layers. DNA profiling of all six phESC lines demonstrates that they are MHC matched with the oocyte donors. The study of imprinted genes demonstrated further evidence of the parthenogenetic origin of the phESC lines. Our research has resulted in a protocol for the production of human parthenogenetic embryos and the derivation of stem cell lines from them, which minimizes the presence of animal-derived components, making the derived phESC lines more suitable for potential clinical use.

We report that angiotensin-converting enzyme (ACE), a critical physiologic regulator of blood pressure, angiogenesis, and inflammation, is a novel marker for identifying hemangioblasts differentiating from human embryonic stem cells (hESC). We demonstrate that ACE ؉ CD45 ؊ CD34 ؉/؊ hemangioblasts are common yolk sac (YS)-like progenitors for not only endothelium but also both primitive and definitive human lymphohematopoietic cells. Thrombopoietin and basic fibroblast growth factor are identified as critical factors for the proliferation of human hemangioblasts.The developmental sequence of human embryoid body hematopoiesis is remarkably congruent to the timeline of normal human YS development, which occurs during weeks 2 to 6 of human gestation. Furthermore, ACE and the reninangiotensin system (RAS) directly regulate hemangioblast expansion and differentiation via signaling through the angiotensin II receptors AGTR1 and AGTR2. ACE enzymatic activity is required for hemangioblast expansion, and differentiation toward either endothelium or multipotent hematopoietic progenitors is dramatically augmented after manipulation of angiotensin II signaling with either AGTR1-or AGTR2-specific inhibitors. The RAS can therefore be exploited to direct the hematopoietic or endothelial fate of hESC-derived hemangioblasts, thus providing novel opportunities for human tissue engineering. Moreover, the initial events of human hematoendotheliogenesis can be delineated in a manner previously impossible because of inaccessibility to early human embryonic tissues. IntroductionHuman hematopoiesis initiates during the third week of development with formation of yolk sac (YS) blood islands derived from extraembryonic mesoderm. 1,2 The YS generates primarily nucleated primitive erythroblasts that express predominantly embryonic hemoglobins (eg, ε 2 2 , Gower I; ␣ 2 ε 2 , Gower II; and 2 ␥ 2 , Portland), and primitive macrophages. 3 After the onset of circulation at approximately 21 days of embryonic development, YS blood cells continue to circulate in embryonic blood, but the fetal liver (FL) eventually replaces the YS as the main hematopoietic organ beginning at 5 to 6 weeks. FL hematopoiesis is dominated by adult-type definitive erythrocytes (enucleated, macrocytic) expressing abundant fetal (HbF; ␣ 2 ␥ 2 ), adult (HbA; ␣ 2 ␤ 2 ) hemoglobins, but limited embryonic hemoglobins. [4][5][6] The FL also produces rare megakaryocytes, granulocytes, macrophages, lymphocytes, and blast cells. 7 Definitive erythroid, myeloid, megakaryocytic, natural killer-B (NK-B) lymphoid, and multipotent progenitors, with limited long-term engraftment potential, are briefly detected during the late YS (eg, 4-5 weeks) stage, before fetal liver hematopoiesis, in both mice and humans. 2,[8][9] The ephemeral nature of definitivetype YS hematopoiesis suggests a gradual yolk sac-fetal liver transition. 1 Indeed, the classic embryonic 3 fetal 3 adult globin switch in early erythrocytes occurs sequentially over time and probably in the same clonal lineage. For examp...

Individual HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines have the potential for cell-based therapy in a significant number of individuals, provided the HLA haplotype is prevalent. We report the successful derivation of four stable hpSC-Hhom lines from both HLA homozygous and HLA heterozygous donors. Of these, the hpSC-Hhom-4 line carries the HLA haplotype found most commonly within the U.S. population, and is shared by different racial groups. These hpSC-Hhom lines demonstrate typical human embryonic stem cell morphology, expressing appropriate stem cell markers and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. G-banded karyotyping demonstrates a normal 46,XX karyotype in lines hpSC-Hhom-1 and hpSC-Hhom-4, and chromosomal anomalies in lines hpSC-Hhom-2 and hpSC-Hhom-3, both derived from the same donor. HLA genotyping of all four hpSC-Hhom lines demonstrates that they are HLA homozygous. Furthermore, in the case of HLA heterozygous donors, the hpSC-Hhom lines inherit the haplotype from only one of the donor's parents. Single-nucleotide polymorphism (SNP) data analysis suggests that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome as assessed by SNP analysis. The protocol used for deriving these HLA homozygous stem cell lines minimizes the use of animal-derived components, which makes them more appealing for potential clinical application.

Correct duplication of stem cell genetic material and its appropriate segregation into daughter cells are requisites for tissue, organ and organism homeostasis. Disruption of stem cell genomic integrity can lead to developmental abnormalities and cancer. Roles of the Smc5/6 structural maintenance of chromosomes complex in pluripotent stem cell genome maintenance have not been investigated, despite its important roles in DNA synthesis, DNA repair and chromosome segregation as evaluated in other model systems. Using mouse embryonic stem cells (mESCs) with a conditional knockout allele of Smc5, we showed that Smc5 protein depletion resulted in destabilization of the Smc5/6 complex, accumulation of cells in G2 phase of the cell cycle and apoptosis. Detailed assessment of mitotic mESCs revealed abnormal condensin distribution and perturbed chromosome segregation, accompanied by irregular spindle morphology, lagging chromosomes and DNA bridges. Mutation of Smc5 resulted in retention of Aurora B kinase and enrichment of condensin on chromosome arms. Furthermore, we observed reduced levels of Polo-like kinase 1 at kinetochores during mitosis. Our study reveals crucial requirements of the Smc5/6 complex during cell cycle progression and for stem cell genome maintenance.