Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages ...of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patient specific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.
Postnatal hematopoietic stem cells (HSCs) from umbilical cord blood and adult marrow/blood have been successfully used for treating various human diseases in the past several decades. However, the ...availability of optimal numbers of HSCs from autologous patients or allogeneic donors with adequate match remains a great barrier to improve and extend HSC and marrow transplantation to more needing patients. In addition, the inability to expand functional human HSCs to sufficient quantity in the laboratory has hindered our research and understanding of human HSCs and hematopoiesis. Recent development in reprogramming technology has provided patient-specific pluripotent stem cells (iPSCs) as a powerful enabling tool for modeling disease and developing therapeutics. Studies have demonstrated the potential of human iPSCs, which can be expanded exponentially and amenable for genome engineering, for using in modeling both inherited and acquired blood diseases. Proof-of-principle studies have also shown the feasibility of iPSCs in gene and cell therapy. Here, we review the recent development in iPSC-based blood disease modeling, and discuss the unsolved issues and challenges in this new and promising field.
Although HMGA1 (high-mobility group A1; formerly HMG-I/Y) is an oncogene that is widely overexpressed in aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only ...beginning to emerge. HMGA1 encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. To determine how HMGA1 leads to neoplastic transformation, we looked for genes regulated by HMGA1 using gene expression profile analysis. Here, we show that the STAT3 gene, which encodes the signaling molecule signal transducer and activator of transcription 3 (STAT3), is a critical downstream target of HMGA1a. STAT3 mRNA and protein are up-regulated in fibroblasts overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a in fibroblasts. HMGA1a also binds directly to a conserved region of the STAT3 promoter in vivo in human leukemia cells by chromatin immunoprecipitation and activates transcription of the STAT3 promoter in transfection experiments. To determine if this pathway contributes to HMGA1-mediated transformation, we investigated STAT3 expression in our HMGA1a transgenic mice, all of which developed aggressive lymphoid malignancy. STAT3 expression was increased in the leukemia cells from our transgenics but not in control cells. Blocking STAT3 function induced apoptosis in the transgenic leukemia cells but not in controls. In primary human leukemia samples, there was a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function in human leukemia or lymphoma cells led to decreased cellular motility and foci formation. Our results show that the HMGA1a-STAT3 axis is a potential Achilles heel that could be exploited therapeutically in hematopoietic and other malignancies overexpressing HMGA1a.
We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured ...under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFi) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.
Differentiated cells derived from pluripotent human embryonic stem (hES) cells offer the opportunity for new transplantation therapies. However, hES cells and their differentiated progeny express ...highly polymorphic MHC molecules that serve as major graft rejection antigens to the immune system of allogeneic hosts. To achieve sustained engraftment of donor cells, strategies must be developed to overcome graft rejection without broadly suppressing host immunity. One approach entails induction of donor-specific immune tolerance by establishing chimeric engraftment in hosts with haemopoietic cells derived from an existing hES cell line. We aimed to develop methods to efficiently differentiate hES cells to haemopoietic cells, including immune-modulating leucocytes, a prerequisite of the tolerance induction strategies applying to hES cell-mediated transplantation.
We developed a method to generate a broad range of haemopoietic cells from hES-generated embryonic bodies in the absence of murine stromal feeder cells. Embryonic bodies were further cultured in the presence of haemopoietic cytokines. In addition to flow cytometric analyses of haemopoietic cell markers, we analysed the hES cell-derived haemopoietic cells by colony-forming assays (for erythroid and myeloid progenitor cells), cytochemical staining, and mixed leucocyte reactions to determine the functional capacity of the generated antigen-presenting cells.
12 independent experiments were done. When selected growth factors were added, leucocytes expressing CD45 were generated and released into culture media for 6–7 weeks. Under the condition used, both erythroid and myeloid progenitor cells were generated. About 25% of the generated leucocytes acquired MHC class II and costimulatory molecule expression. These hES-derived, MHC class II+ leucocytes resembled dendritic cells and macrophages, and they functioned as antigen-presenting cells capable of eliciting allogeneic CD4 and CD8 T-cell responses in culture.
The hES cell-derived antigen-presenting cells could be used to regulate alloreactive T cells and induce immune tolerance for improvement of the transplant acceptance of hES-cell derivatives.
Traditional methods of dividing petroleum reservoirs are inefficient, and the accuracy of onehidden-layer BP neural network is not ideal when applied to dividing reservoirs. This paper proposes to ...use the deep learning models to solve the reservoir division problem. We apply multiple-hidden-layer BP neural network and convolutional neural network models, and adjust the network structures according to the characteristics of the reservoir problem. The results show that the deep learning models are better than onehidden- layer BP neural network, and the performance of the convolutional neural network is very close to the artificial work.
We report here a lentiviral vector system for regulated transgene expression. We used the tetracycline repressor fused with a transcriptional suppression domain (tTS) to specifically suppress ...transgene expression. Human cells were first transduced with a tTS-expressing vector and subsequently transduced with a second lentiviral vector-containing transgene controlled by a regular promoter adjacent to a high-affinity tTS-binding site (tetO). After optimizing the location of the tetO site in the latter vector, we achieved a better inducible transgene expression than the previous lentiviral vectors using the tetracycline repressor systems. In this new system, the transgene transcription from a cellular promoter such as EF1alpha or ubiquitin-C promoter is suppressed by the tTS bound to the nearby tetO site. In the presence of the tetracycline analog doxycycline (Dox), however, the tTS binding is released from the transgene vector and transcription from the promoter is restored. Thus, this system simply adds an extra level of regulation, suitable for any types of promoters (ubiquitous or cell-specific). We tested this tTS-suppressive, Dox-inducible system in 293T cells, human multipotent hematopoietic progenitor cells, and three human embryonic stem cell lines, using a dual-gene vector containing the green fluorescent protein reporter or a cellular gene. We observed a tight suppression in the uninduced state. However, the suppression is reversible, and transgene expression was restored at 5 ng/ml Dox. The lentiviral vectors containing the tTS-suppressive, Dox-inducible system offer a universal, inducible, and reversible transgene expression system in essentially any mammalian cell types, including human embryonic stem cells.
Using PIG‐A gene targeting and an inducible PIG‐A expression system, this study established a conditional PIG‐A knockout model in human induced pluripotent stem cells that allowed for the production ...of glycosylphosphatidylinositol‐anchored protein (GPI‐AP)‐deficient blood cells. This conditional PIG‐A knockout model should be a valuable tool for studying the importance of GPI‐APs in hematopoiesis and human development.
PIG‐A is an X‐linked gene required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; thus, PIG‐A mutant cells have a deficiency or absence of all GPI‐anchored proteins (GPI‐APs). Acquired mutations in hematopoietic stem cells result in the disease paroxysmal nocturnal hemoglobinuria, and hypomorphic germline PIG‐A mutations lead to severe developmental abnormalities, seizures, and early death. Human induced pluripotent stem cells (iPSCs) can differentiate into cell types derived from all three germ layers, providing a novel developmental system for modeling human diseases. Using PIG‐A gene targeting and an inducible PIG‐A expression system, we have established, for the first time, a conditional PIG‐A knockout model in human iPSCs that allows for the production of GPI‐AP‐deficient blood cells. PIG‐A‐null iPSCs were unable to generate hematopoietic cells or any cells expressing the CD34 marker and were defective in generating mesodermal cells expressing KDR/VEGFR2 (kinase insert domain receptor) and CD56 markers. In addition, PIG‐A‐null iPSCs had a block in embryonic development prior to mesoderm differentiation that appears to be due to defective signaling through bone morphogenetic protein 4. However, early inducible PIG‐A transgene expression allowed for the generation of GPI‐AP‐deficient blood cells. This conditional PIG‐A knockout model should be a valuable tool for studying the importance of GPI‐APs in hematopoiesis and human development.
Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival ...of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.
Human induced pluripotent stem (iPS) cells have been generated from various cell types including blood cells, and offer certain advantages as a starting population for reprogramming postnatal somatic ...cells. Unlike adult stem cells, iPS cells can proliferate limitlessly in culture while retaining their potential to differentiate into any cell type, including hematopoietic lineages. Derivation of patient-specific iPS cells, in combination with improved hematopoietic differentiation protocols, provides an alternative to generate histocompatible stem cells for bone marrow transplantation. In addition, the ability to reprogram blood cells and redifferentiate iPS cells back to hematopoietic lineages provides opportunities to establish novel models for acquired and inherited blood diseases. This article will summarize recent progress in human iPS cells derived from blood cells and hematopoietic differentiation from iPS cells. Advantages of blood as a source for reprogramming and applications in regenerative medicine will be discussed.
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