诱导多能干细胞与心脏再生

诱导多能干细胞(induced pluripotent stem cells, iPSCs)研究的快速发展为心血管转化医学研究领域提供了新的策略. 诱导多能干细胞不仅具有与胚胎干细胞类似的多能性, 且巧妙地回避了胚胎干细胞面临的伦理学问题和免疫排斥反应. 心肌细胞等成体心血管细胞在发生心血管疾病后增殖能力有限, 而iPSCs 来源的心血管细胞在心脏再生治疗中颇具应用前景,是理想的细胞来源, 因此在基础医学和转化医学研究领域受到广泛关注. 就iPSCs 的发展过程及其在心脏再生中的应用作一综述, 并探讨目前iPSCs 在心脏再生临床转化中亟待解决的问题.     

Comparative pluripotency analysis of mouse embryonic stem cells derived from wild-type and infertile hermaphrodite somatic cell nuclear transfer blastocysts

Therapeutic cloning, whereby embryonic stem cells （ESCs） are derived from patient-specific cloned blastocysts via somatic cell nuclear transfer （SCNT）, holds great promise for treating many human diseases using regenerative medicine. Teratoma formation and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells （hESCs） have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmis- sion, which would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines lack full multi- or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3 genetically defective nuclear transfer- ESC （ntESC） lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted via the germ line, in contrast with normal ntESCs. Our results in- dicate that teratoma formation assays with hESCs might be an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards are required to assess the safety of hESCs for therapeutic cloning.     

Application of Induced Pluripotent Stem Cells Derived from Human Periodontal Ligament Cells in Bone Regeneration

The purpose of this study was to primarily culture human periodontal ligament cells(hPDLCs) and to reprogram hPDLCs with exogenous genes via a lentivirus-mediated transfection system. Then induced pluripotent stem cells derived from h PDLCs(hPDLC-iPSCs) were identified. Alizarin red staining was used to observe the formation of mineralized nodules and real-time Polymerase Chain Reaction(PCR) was used to detect the expression of osteogenic genes. For the in vivo experiment, nude mouse skull defect models were established and cell sheets were made to repair the bone defect. The reprogrammed cells were positive for alkaline phosphatase(ALP) staining and embryonic stem cells(ESCs)-specific proteins, and could form teratomas. After osteogenic induction, alizarin red staining showed that the number of mineralized nodules in the h PDLC-i PSCs group was more and the osteogenic related factors ALP, osteocalcin(OCN), Col-I and Runx2 were also expressed higher in hPDLC-iPSCs. The hPDLC-iPSC cell sheets were all successfully made. Histological analysis showed that the h PDLC-i PSC cell sheet got new bone formation. These results demonstrated that hPDLC-iPSCs were successfully generated from human periodontal ligament fibroblasts and hPDLC-iPSC cell sheets provided new options for bone tissue engineering.     

Monoclonal mice generated by nuclear transfer from mature B and T donor cells

Cloning from somatic cells is inefficient, with most clones dying during gestation. Cloning from embryonic stem (ES) cells is much more effective, suggesting that the nucleus of an embryonic cell is easier to reprogram. It is thus possible that most surviving clones are, in fact, derived from the nuclei of rare somatic stem cells present in adult tissues, rather than from the nuclei of differentiated cells, as has been assumed. Here we report the generation of monoclonal mice by nuclear transfer from mature lymphocytes. In a modified two-step cloning procedure, we established ES cells from cloned blastocysts and injected them into tetraploid blastocysts to generate mice. In this approach, the embryo is derived from the ES cells and the extra-embryonic tissues from the tetraploid host. Animals cloned from a B-cell nucleus were viable and carried fully rearranged immunoglobulin alleles in all tissues. Similarly, a mouse cloned from a T-cell nucleus carried rearranged T-cell-receptor genes in all tissues. This is an unequivocal demonstration that a terminally differentiated cell can be reprogrammed to produce an adult cloned animal.     

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.     

Sporadic immunogenic tumours avoid destruction by inducing T-cell tolerance

The recognition and elimination of tumours by T cells, a process termed cancer immunosurveillance, is effective against certain virus-associated cancers. Spontaneous tumours often induce a specific immune response and are therefore also immunogenic. However, it is not clear whether they can be controlled by T cells. The immunosurveillance hypothesis postulates that tumours, if they eventually grow, escaped T-cell recognition by losing immunogenicity. Here we show, by generating a mouse model of sporadic cancer based on rare spontaneous activation of a dormant oncogene, that immunogenic tumours do not escape their recognition but induce tolerance. In this model, tumours derive from single cells and express a tumour-specific transplantation rejection antigen. Whereas vaccinated mice remain tumour-free throughout their lifetime, naive mice always develop a progressively growing tumour. We also show that despite specific recognition by T cells, the tumours do not lose their intrinsic immunogenicity and are rejected after transplantation in T-cell-competent recipients. Furthermore, in the primary host tumour-induced tolerance is associated with the expansion of non-functional T cells. Together, our data argue against immunosurveillance of spontaneous cancer.     

Mice cloned from olfactory sensory neurons

Cloning by nuclear transplantation has been successfully carried out in various mammals, including mice. Until now mice have not been cloned from post-mitotic cells such as neurons. Here, we have generated fertile mouse clones derived by transferring the nuclei of post-mitotic, olfactory sensory neurons into oocytes. These results indicate that the genome of a post-mitotic, terminally differentiated neuron can re-enter the cell cycle and be reprogrammed to a state of totipotency after nuclear transfer. Moreover, the pattern of odorant receptor gene expression and the organization of odorant receptor genes in cloned mice was indistinguishable from wild-type animals, indicating that irreversible changes to the DNA of olfactory neurons do not accompany receptor gene choice.     

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.     

Human embryonic stem cell lines derived from single blastomeres

The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. In this proof-of-principle study, multiple biopsies were taken from each embryo using micromanipulation techniques and none of the biopsied embryos were allowed to develop in culture. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos.     

Generation of tetraploid complementation mice from embryonic stem cells cultured with chemical defined medium

Mouse embryonic stem cells(mESCs)derived from inner cell mass(ICM)of pre-implantation embryos,can maintain undifferentiated state when cultured in N2B27 medium supplemented with GSK3inhibitor CHIR99021 and MEK inhibitor PD0325901(‘‘2i’’)and leukemia inhibitor factor(LIF).Compare to conventional culture medium,all components of this medium are defined.With the N2B27 medium,‘‘2i’’and LIF,mESCs can contribute to the germline of the chimeric embryos,however,whether the‘‘all-ES cells’’mice can been generated by tetraploid complementation is unclear yet,while the tetraploid complementation serve as a golden standard to assess the pluripotency of ES cells.Here,our study showed that mESCs derived and cultured with the N2B27 complete medium could generate fertile mice by tetraploid complementation.In addition,the survival rate of tetraploid complementation mice produced by inbred mES cell lines is higher than the conventional culture condition,and increased the percentage of Oct4 positive cells contrast to conventional medium either.Therefore,the N2B27 medium supplemented with‘‘2i’’and LIF is an alternative choice forthe derivation and long-term culture of mouse embryonic stem cells.     

Depletion of the predominant B-cell population in immunoglobulin mu heavy-chain transgenic mice

The transgenic mouse line M54 was generated by introducing a functionally-rearranged immunoglobulin mu heavy-chain gene into the germ line of a C57B1/6 inbred mouse. Previous examination of the antibodies produced by B-cell hybridomas derived from transgenic M54 mice showed that the presence of the mu transgene grossly altered the immunoglobulin repertoire of unimmunized animals, suggesting that these mice suffer from a serious immunoregulatory perturbation. Studies presented here introduce a new perspective on this functional defect. We show that the lymphoid tissues from these transgenic mice lack virtually all conventional bone-marrow-derived B cells, which constitute the predominant B-cell population in normal mice and which typically produce primary and secondary antibody responses to T-cell-dependent antigens. Moreover, the bone marrow from transgenic M54 mice is depleted of pre-B lymphocytes, indicating a serious defect in early B-cell lymphopoiesis. In contrast, CD5 (Ly-1) B cells, a second B-cell population displaying a characteristic set of cell surface markers which are derived from distinct precursors in the peritoneum, are represented at normal frequencies in these transgenic mice. Thus, the presence of the rearranged immunoglobulin heavy-chain transgene in M54 mice results in an unexpected selective developmental defect that impairs the development of bone-marrow-derived pre-B and B cells without affecting Ly-1 B cells.     

Analysis of T-cell subsets in rejection of Kb mutant skin allografts differing at class I MHC

The T-cell subpopulations which initiate and mediate tissue allograft rejection remain controversial. In the present study we attempted to identify the phenotype and function of the T-cell subset(s) primarily responsible for the rejection of skin allografts differing at a single class I locus in the major histocompatibility complex (MHC). We found that the rejection rates by B6 mice (H-2b) of four different class I mutant (Kbm) skin allografts form a distinct hierarchy. This hierarchy correlates strikingly and uniquely with the relative precursor frequencies of Lyt2+ interleukin-2-secreting T-helper cells reactive against the various Kbm mutants. To investigate the role of Lyt2+ T cells in the rejection of class I-disparate skin allografts directly, H-2b nude mice were engrafted with Kbm skin allografts and then reconstituted with L3T4+ or Lyt2+ T-cell subpopulations from syngeneic H-2b mice. Lyt2+ T cells were observed to be both necessary and sufficient for the rejection of class I-disparate Kbm skin allografts, whereas L3T4+ T cells were neither necessary nor sufficient. These results identify the Lyt2+ interleukin-2-secreting T-cell subset as the critical cell type determining the rejection rate of class I-disparate Kbm skin allografts.     

Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.     

Pluripotency of spermatogonial stem cells from adult mouse testis

Embryonic germ cells as well as germline stem cells from neonatal mouse testis are pluripotent and have differentiation potential similar to embryonic stem cells, suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male. Here we show the isolation of SSCs from adult mouse testis using genetic selection, with a success rate of 27%. These isolated SSCs respond to culture conditions and acquire embryonic stem cell properties. We name these cells multipotent adult germline stem cells (maGSCs). They are able to spontaneously differentiate into derivatives of the three embryonic germ layers in vitro and generate teratomas in immunodeficient mice. When injected into an early blastocyst, SSCs contribute to the development of various organs and show germline transmission. Thus, the capacity to form multipotent cells persists in adult mouse testis. Establishment of human maGSCs from testicular biopsies may allow individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells. Furthermore, these cells may provide new opportunities to study genetic diseases in various cell lineages.     

空间环境对黑色素瘤B16细胞体内增殖及免疫原性的影响

初步探讨空间环境对黑色素瘤B16细胞增殖及免疫原性的影响。选择4株经体外实验筛选出生物学性状变异明显的第20颗返回式卫星搭载的空间培养B16细胞,检测其体内增殖能力及免疫原性的变化。将筛选出的4株空间培养B16细胞接种于C57BL/6小鼠,其中一组接种于腹腔,观察荷瘤小鼠生存期;另一组接种于腋下皮肤,检测小鼠出瘤时间。接种至2周时,处死,分离荷瘤小鼠移植瘤和血清。称重荷瘤小鼠移植瘤,观察空间培养B16细胞增殖能力;利用HE染色法观察荷瘤小鼠移植瘤切片细胞形态;采用放射免疫分析法(Radioimmunoassay,RIA)测定荷瘤小鼠血清白介素-2(InterLeukin-2,IL-2)浓度,观察空间培养B16细胞免疫原性变化情况。与对照细胞相比,1株空间培养B16细胞的荷瘤小鼠生存期明显缩短,出瘤时间明显延长。HE结果显示:此株空间培养B16肿瘤组织内有淋巴细胞浸润;RIA结果显示:3株空间培养B16细胞荷瘤小鼠血清IL-2浓度明显增加。空间环境的复合因素可诱导B16细胞增殖能力及免疫原性产生变化。     

Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance

Allogeneic reactions are the major limitation to organ transplantation. These are manifested as rejection of the grafted tissue, and also, in the case of bone marrow transplantation (BMT), graft-versus-host disease (GVHD). Recent methods of avoiding GVHD, by depleting T cells from donor marrow, have led to an increased incidence of marrow graft rejection. Current recipient conditioning protocols involving drugs or irradiation cannot safely be increased, so alternatives must be found. Monoclonal antibodies can be used to control immune responses in vivo, and would be useful in this context if we could define and deplete the cells responsible for marrow rejection. We show here that elimination of residual L3T4+ and Lyt-2+ cells from mice receiving fully mismatched bone marrow abrogates rejection and promotes tolerance to donor-type skin grafts, even in sub-lethally irradiated recipients.