技术资料

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However,the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However,5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood,as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted,mediated by CD8(+) lymphocytes,and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959) View Publication

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) can differentiate into many cell types and are important for regenerative medicine; however,further work is needed to reliably differentiate hESC and hiPSC into neural-restricted multipotent derivatives or specialized cell types under conditions that are free from animal products. Toward this goal,we tested the transition of hESC and hiPSC lines onto xeno-free (XF) / feeder-free conditions and evaluated XF substrate preference,pluripotency,and karyotype. Critically,XF transitioned H9 hESC,Shef4 hESC,and iPS6-9 retained pluripotency (Oct-4 and NANOG),proliferation (MKI67 and PCNA),and normal karyotype. Subsequently,XF transitioned hESC and hiPSC were induced with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to generate neuralized spheres containing primitive neural precursors,which could differentiate into astrocytes and neurons,but not oligoprogenitors. Further neuralization of spheres via LIF supplementation and attachment selection on CELLstart substrate generated adherent human neural stem cells (hNSC) with normal karyotype and high proliferation potential under XF conditions. Interestingly,adherent hNSC derived from H9,Shef4,and iPS6-9 differentiated into significant numbers of O4+ oligoprogenitors (∼20-30%) with robust proliferation; however,very few GalC+ cells were observed (∼2-4%),indicative of early oligodendrocytic lineage commitment. Overall,these data demonstrate the transition of multiple hESC and hiPSC lines onto XF substrate and media conditions,and a reproducible neuralization method that generated neural derivatives with multipotent cell fate potential and normal karyotype. View Publication

An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion and folding in an in vitro model of human corticogenesis. Deletion of PTEN stimulates proliferation and generates significantly larger and substantially folded cerebral organoids. This genetic modification allows sustained cell cycle re-entry,expansion of the progenitor population,and delayed neuronal differentiation,all key features of the developing human cortex. In contrast,Pten deletion in mouse organoids does not lead to folding. Finally,we utilized the expanded cerebral organoids to show that infection with Zika virus impairs cortical growth and folding. Our study provides new insights into the mechanisms regulating the structure and organization of the human cortex. View Publication

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways,however,it remains unclear as to what extent these pathways can be exploited,either individually or in combination,in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro,but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways,we developed a high-density microbioreactor array (HDMA) - a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt,Hedgehog,IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation,inducing cell cycle activity marked by Ki67,and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration. View Publication

Somatic cell nuclear transfer,cell fusion,or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors,Brn2 (also known as Pou3f2),Ascl1 and Myt1l,can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1,these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers,even after downregulation of the exogenous transcription factors. Importantly,the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells,as well as pluripotent stem cells,can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine. View Publication

Gastric cancer (GC) is one of the most common human cancers. Spheroid colony formation is an effective model for characterization of cancer stem cells. However,gene expression profiles of spheroid colonies obtained from GC cells have not been examined. We performed microarray analyses by Human Genome U133 Plus 2.0 Array in spheroid body-forming and parental cells from MKN-45 and MKN-74 GC cell lines. Kinesin family member C1 (KIFC1) was expressed textgreater2-fold higher in spheroid body-forming cells than in parental cells in both GC lines. Both the number and size of spheres from MKN-45 cells were significantly reduced upon KIFC1 siRNA-transfection compared with negative control siRNA-transfection. Immunohistochemical analysis of 114 GC tissue samples revealed that 42 (37%) of GC cases were positive for KIFC1 expression. GC cases positive for KIFC1 were found more frequently in stage III/IV cases than in stage I/II cases. GC cases positive for KIFC1 were found more frequently in intestinal type GC cases than in diffuse type GC cases. Furthermore,KIFC1-positive GC cases showed high Ki-67 labeling index. Kaplan-Meier analysis demonstrated that KIFC1 expression was not associated with survival. We found positive expression of KIFC1 in CD44‑positive GC and aldehyde dehydrogenase 1 (ALDH1)-positive GC cells. Our results showed that KIFC1 is overexpressed in GC. Since knockdown of KIFC1 inhibited sphere formation,KIFC1 likely plays an important role in cancer stem cells. View Publication

Reprogramming of a limited number of human cell types has been achieved through ectopic expression of four transcription factors to yield induced pluripotent stem (iPS) cells that closely resemble human embryonic stem cells (ESCs). Here,we determined functional and epigenetic properties of iPS cells generated from human umbilical vein endothelial cells (HUVEC) by conventional method of direct reprogramming. Retroviral overexpression of four transcription factors resets HUVEC to the pluripotency. Human endothelial cell-derived iPS (endo-iPS) cells were similar to human ESCs in morphology,gene expression,in vitro and in vivo differentiation capacity. Endo-iPS cells were efficiently differentiated in vitro into endothelial cells. Using genome-wide methylation profiling we show that promoter elements of endothelial specific genes were methylated following reprogramming whereas pluripotency-related gene promoters were hypomethylated similar to levels observed in ESCs. Genome-wide methylation analysis of CpG sites located in the functional regions of over than 14,000 genes indicated that human endo-iPS cells were highly similar to human ES cells,although differences in methylation levels of 46 genes were found. Overall CpG methylation of promoter regions in the pluripotent cells was higher than in somatic. We also show that during reprogramming female human endo-iPS cells exhibited reactivation of the somatically silenced X chromosome. Our findings demonstrate that iPS cells can be generated from human endothelial cells and reprogramming resets epigenetic status of endothelial cells to pluripotency. View Publication

During mammalian embryonic development,the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors,such as OCT3/4,SOX2,KLF4 and c-MYC (hereafter referred to as OSKM),albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells,intermediate cells transiently show gene expression profiles resembling mesendoderm,which is a major component of the primitive streak. Based on these findings,we discover that forkhead box H1 (FOXH1),a transcription factor required for anterior primitive streak specification during early development,significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation,including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process,human somatic cells go through a transient state that resembles mesendoderm. View Publication

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells,capable of germline transmission,from mouse somatic cells by transduction of four defined transcription factors. Here,we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4,and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology,proliferation,surface antigens,gene expression,epigenetic status of pluripotent cell-specific genes,and telomerase activity. Furthermore,these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts. View Publication

Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However,such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions,mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic,neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+,clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin,and on maturation,differentially stain positive for neuronal,glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers,are negative for major histocompatibility complex (MHC) class I and II antigens,transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but,in any case,could have profound clinical and pharmacological implications. Finally,the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure. View Publication

In a patient with refractory anemia with excess blasts (RAEB),a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly,the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However,PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays,CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis,thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient. View Publication

Although there is evidence that endothelial cells are important targets for human pathogenic Bartonella species,the primary niche of infection is unknown. Here we elucidated whether human CD34+ hematopoietic progenitor cells (HPCs) internalize B. henselae and may serve as a potential niche of the pathogen. We showed that B. henselae does not adhere to or invade human erythrocytes. In contrast,B. henselae invades and persists in HPCs as shown by gentamicin protection assays,confocal laser scanning microscopy (CLSM),and electron microscopy (EM). Fluorescence-activated cell sorting (FACS) analysis of glycophorin A expression revealed that erythroid differentiation of HPCs was unaffected following infection with B. henselae. The number of intracellular B. henselae continuously increased over a 13-day period. When HPCs were infected with B. henselae immediately after isolation,intracellular bacteria were subsequently detectable in differentiated erythroid cells on day 9 and day 13 after infection,as shown by CLSM,EM,and FACS analysis. Our data provide,for the first time,evidence that a bacterial pathogen is able to infect and persist in differentiating HPCs,and suggest that HPCs might serve as a potential primary niche in Bartonella infections. View Publication