技术资料

The clinical use of human embryonic stem cells (hESCs) requires efficient cellular expansion that must be paired with an ability to generate specialized progeny through differentiation. Self-renewal and differentiation are deemed inherent hallmarks of hESCs and a growing body of evidence suggests that initial culture conditions dictate these two aspects of hESC behavior. Here,we reveal that defined culture conditions using commercial mTeSR1 media augment the expansion of hESCs and enhance their capacity for neural differentiation at the expense of hematopoietic lineage competency without affecting pluripotency. This culture-induced modification was shown to be reversible,as culture in mouse embryonic fibroblast-conditioned media (MEF-CM) in subsequent passages allowed mTeSR1-expanded hESCs to re-establish hematopoietic differentiation potential. Optimal yield of hematopoietic cells can be achieved by expansion in mTeSR1 followed by a recovery period in MEF-CM. Furthermore,the lineage propensity to hematopoietic and neural cell types could be predicted via analysis of surrogate markers expressed by hESCs cultured in mTeSR1 versus MEF-CM,thereby circumventing laborious in vitro differentiation assays. Our study reveals that hESCs exist in a range of functional states and balance expansion with differentiation potential,which can be modulated by culture conditions in a predictive and quantitative manner. Stem Cells 2015;33:1142-1152. View Publication

The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However,it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps,we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in beta-hemoglobin gene (HBB) that cause severe beta-thalassemia (beta-Thal),corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting,and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing,we uncovered seven copy number variations,five small insertions/deletions,and 64 single nucleotide variations (SNVs) in beta-Thal iPSCs before the gene targeting step and found a single small copy number variation,19 insertions/deletions,and 340 single nucleotide variations in the final gene-corrected beta-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps,suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting. View Publication

BACKGROUND Humanized mouse models are still under development,and various protocols exist to improve human cell engraftment and function. METHODS Fourteen NOD/SCID/IL-2Rγnull (NSG) mice (4‒5 wk old) were conditioned with busulfan and injected with human umbilical cord blood (hUCB)-derived CD34(+) hematopoietic stem cells (HSC) via retro-orbital sinuses. The bone marrow (BM),spleen,and peripheral blood (PB) were analyzed 8 and 12 weeks after HSC transplantation. RESULTS Most of the NSG mice tolerated the regimen well. The percentage of hCD45(+) and CD19(+) cells rose significantly in a time-dependent manner. The median percentage of hCD45(+)cells in the BM was 55.5% at week 8,and 67.2% at week 12. The median percentage of hCD45(+) cells in the spleen at weeks 8 and 12 was 42% and 51%,respectively. The median percentage of hCD19(+) cells in BM at weeks 8 and 12 was 21.5% and 39%,respectively (P=0.04). Similarly,the median percentage of hCD19(+) cells in the spleen at weeks 8 and 12 was 10% and 24%,respectively (P=0.04). The percentage of hCD19(+) B cells in PB was 23% at week 12. At week 8,hCD3(+) T cells were barely detectable,while hCD7(+) was detected in the BM and spleen. The percentage of hCD3(+) T cells was 2‒3% at week 12 in the BM,spleen,and PB of humanized NSG mice. CONCLUSION We adopted a simplified protocol for establishing humanized NSG mice. We observed a higher engraftment rate of human CD45(+) cells than earlier studies without any significant toxicity. And human CD45(+) cell engraftment at week 8 was comparable to that of week 12. View Publication

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However,little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb),97A6,that specifically detects human Ba,MC (lung,skin),and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils,eosinophils,monocytes,lymphocytes,platelets) did not react with MoAb 97A6,and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (textgreater98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40),Ba-eosinophil colonies (7 of 40),Ba-macrophage colonies (3 of 40),and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast,no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells,starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (textgreater30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors,MC progenitors,Ba progenitors,as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors. View Publication

To study molecular events involved in B lymphocyte development and V(D)J rearrangement,we have established an efficient system for the differentiation of embryonic stem (ES) cells into mature Ig-secreting B lymphocytes. Here,we show that B lineage cells generated in vitro from ES cells are functionally analogous to normal fetal liver-derived or bone marrow-derived B lineage cells at three important developmental stages: first,they respond to Flt-3 ligand during an early lymphopoietic progenitor stage; second,they become targets for Abelson murine leukemia virus (A-MuLV) infection at a pre-B cell stage; third,they secrete Ig upon stimulation with lipopolysaccharide at a mature mitogen-responsive stage. Moreover,the ES cell-derived A-MuLV-transformed pre-B (EAB) cells are phenotypically and functionally indistinguishable from standard A-MuLV-transformed pre-B cells derived from infection of mouse fetal liver or bone marrow. Notably,EAB cells possess functional V(D)J recombinase activity. In particular,the generation of A-MuLV transformants from ES cells will provide an advantageous system to investigate genetic modifications that will help to elucidate molecular mechanisms in V(D)J recombination and in A-MuLV-mediated transformation. View Publication

Under appropriate conditions in culture,embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic,endothelial,muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo,indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development. View Publication

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs),and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the $\beta$-globin gene (HBB). Sickle hemoglobin damages erythrocytes,causing vasoocclusion,severe pain,progressive organ damage,and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA,together with a single-stranded DNA oligonucleotide donor (ssODN),to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice,ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing,enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells,and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases. View Publication