Advances in application of induced pluripotent stem cells in childhood genetic diseases / 中国生物制品学杂志

@#Somatic cell reprogramming has developed rapidly in the field of modern biology. Induced pluripotent stem cells(iPSCs)obtained through somatic cell reprogramming are not only capable of self-renewal,but also have multidirectional differentiation potential,which plays an important role in disease modeling and regenerative medicine. This paper reviewed the gene reprogramming technology,the disease models of iPSCs and the application prospects of iPSCs in childhood genetic diseases,so as to provide a reference for the application of iPSCs in the research of mechanism and treatment of various genetic diseases.

Development of porcine induced pluripotent stem cells with a CD163 reporter system / 生物工程学报

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

Quantitative proteomics revealed extensive microenvironmental changes after stem cell transplantation in ischemic stroke / 医学前沿

The local microenvironment is essential to stem cell-based therapy for ischemic stroke, and spatiotemporal changes of the microenvironment in the pathological process provide vital clues for understanding the therapeutic mechanisms. However, relevant studies on microenvironmental changes were mainly confined in the acute phase of stroke, and long-term changes remain unclear. This study aimed to investigate the microenvironmental changes in the subacute and chronic phases of ischemic stroke after stem cell transplantation. Herein, induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) were transplanted into the ischemic brain established by middle cerebral artery occlusion surgery. Positron emission tomography imaging and neurological tests were applied to evaluate the metabolic and neurofunctional alterations of rats transplanted with stem cells. Quantitative proteomics was employed to investigate the protein expression profiles in iPSCs-transplanted brain in the subacute and chronic phases of stroke. Compared with NSCs-transplanted rats, significantly increased glucose metabolism and neurofunctional scores were observed in iPSCs-transplanted rats. Subsequent proteomic data of iPSCs-transplanted rats identified a total of 39 differentially expressed proteins in the subacute and chronic phases, which are involved in various ischemic stroke-related biological processes, including neuronal survival, axonal remodeling, antioxidative stress, and mitochondrial function restoration. Taken together, our study indicated that iPSCs have a positive therapeutic effect in ischemic stroke and emphasized the wide-ranging microenvironmental changes in the subacute and chronic phases.

Knockdown of Homeobox D12 Inhibits 7 Factor-Induced Somatic Cell Reprogramming / 中国生物化学与分子生物学报

Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by overexpressing defined transcription factors. The process of reprogramming requires the interaction of various transcription factors to regulate the transformation of cell fate. Hoxd12 (Homeobox D12) is one of the transcription factors regulating the embryonic development of vertebrates, and it plays an outstanding role in the development of the limb, body axis formation, and cell signal transduction. However, any roles of Hoxd12 may play in the somatic cell reprogramming and the pluripotency of embryonic stem cells (ESCs) have not been reported. In this study, we firstly used 7 factors (Sall4-Esrrb-Jdp2-Glis1-Mkk6-Nanog-Kdm2b) and Yamanaka factors (Oct4-Klf4-Sox2) as the research model, combined with RNA interference (shRNA) and gene overexpression, to explore the mechanism of Hoxd12 in somatic cell reprogramming. Moreover, we used CRISPR/Cas9 gene editing to construct Hoxd12 knockout embryonic stem cell lines, and combined embryoid body formation (EB) and RNA sequencing (RNA-seq) to explore the function of Hoxd12 in the pluripotency of ESCs. The conclusions are as follows: (1) Knocking down of Hoxd12 inhibits 7 factor-induced reprogramming (

Induction and differentiation of induced pluripotent stem cells into macrophages: a review / 生物工程学报

Induced pluripotent stem cells (iPSCs) are a type of cells similar to embryonic stem cells but produced by reprogramed somatic cells. Through in vitro differentiation of iPSCs, we can interrogate the evolution history as well as the various characteristics of macrophages. iPSCs derived macrophages are not only a good model for drug screening, but also an important approach for immunotherapy. This review summarizes the advances, challenges, and future directions in the field of iPSCs-derived macrophages.

Targeted elimination of mutant mitochondrial DNA in MELAS-iPSCs by mitoTALENs

Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). We engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) and successfully eliminated the m.3243A>G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Furthermore, we successfully achieved reduction in the human m.3243A>G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

Current challenges in the therapeutic use of induced pluripotent stem cells (iPSCs) in cancer therapy

Induced Pluripotent Stem Cells (iPSCs) technology has catapulted the field of stem-cell biology through ectopic expression of reprogramming factors. Ever since its discovery, the potential of iPSCs has been explored by many scientists to unravel the molecular mechanism responsible for cancer initiation and progression. Besides modeling cancer, the further applications of this technology includes high-throughput drug screening, epigenetic reprogramming of cancer cell state to normal, immunotherapy and regenerative cell therapies. Here, we review the current challenges on clinical applications of iPSCs with respect to understanding cancer and personalizing treatment for the disease (AU)

Development on neurodegenerative disorder modeling with induced pluripotent stem cells / 中国药理学通报

Remarkable advances in cellular reprogramming have made it possible to investigate relevant cell populations derived from induced pluripotent stem cells ( iPSCs ) of patients. Be-cause many diseases have its specific genetic information, using the cells to convert into iPSCs can build up a set of genetic pro-file of diseases. The iPSCs which contain the genetic contribution of the donor can be expanded and differentiated into cells of the affected lineages to show aberrant phenotypes in culture. To date, over fifty such disease models have been reported, and while the field is young and hurdles remain, we can foresee the huge potential of it in drug screening. Recent studies using iP-SCs to model various neurogenetic disorders are summarized. Compared to the traditional methods, we analyze the future de-velopment of iPSC based disease models and its past application on high-throughput screening ( HTS) and high-content screening ( HCS) .

In Vivo Roles of a Patient-Derived Induced Pluripotent Stem Cell Line (HD72-iPSC) in the YAC128 Model of Huntington's Disease

Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients can provide immense opportunities to model human diseases, which may lead to develop novel therapeutics. Huntington's disease (HD) is a devastating neurodegenerative genetic disease, with no available therapeutic options at the moment. We recently reported the characteristics of a HD patient-derived iPSC carrying 72 CAG repeats (HD72-iPSC). In this study, we investigated the in vivo roles of HD72-iPSC in the YAC128 transgenic mice, a commonly used HD mouse model carrying 128 CAG repeats. To do this, we transplanted HD72-iPSC-derived neural precursors into the striatum of YAC128 mice bilaterally and observed a significant behavioral improvement in the grafted mice. Interestingly, the transplanted HD72-iPSC-derived neural precursors formed GABAeric neurons efficiently, but no EM48-positive protein aggregates were detected at 12 weeks after transplantation. Taken together, these results indicate no HD pathology was developed from the grafted cells, or no transmission of HD pathology from the host to the graft occurred at 12 weeks post-transplantation.

Derivation, characterization and retinal differentiation of induced pluripotent stem cells.

Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.

Neuronal Differentiation of a Human Induced Pluripotent Stem Cell Line (FS-1) Derived from Newborn Foreskin Fibroblasts

Isolation of induced pluripotent stem cells (iPSCs) from fully differentiated somatic cells has revolutionized existing concepts of cell differentiation and stem cells. Importantly, iPSCs generated from somatic cells of patients can be used to model different types of human diseases. They may also serve as autologous cell sources that can be used in transplantation therapy. In this study, we investigated the neuronal properties of an iPSC line that is derived from human neonatal foreskin fibroblasts (FS-1). We initially examined the morphology and marker expression of FS-1 cells at undifferentiated stage. We then spontaneously differentiated FS-1 cells in suspension culture and examined the expression of markers representing three germ layers. We finally differentiated FS-1 cells into neuronal lineages by co-culturing them with PA6 stromal cells, and found that, under the conditions we used, they have a tendency to differentiate into more forebrain-type neurons, suggesting that FS-1 iPSC-derived neural cells will be useful to be used in cell therapy of stroke or Huntington's disease, among others. Taken together, FS-1 cells derived from human neonatal fibroblasts exhibit very similar properties with human ES cells, and can provide useful sources for cell therapy and various other applications.

Effects and mechanism of PNS on synaptic transmission in hippocampal CA1 region of rat / 中草药

Objective To investigate the effects of Panax notoginseng saponins(PNS)on both the excitatory and inhibitory synaptic transmission in the pyramidal neurons in hippocampal CA1 region of rats.Methods Wistar male rats(3—4 weeks)were killed by cervical dislocation and hippocampal slices(400 ?m)were prepared,blind whole-cell voltage-clamp recordings were performed on the CA1 pyramidal cells in hippocampal slices to examine and analyze the effects of PNS(0.05—0.4 g/L)on CA1 afferent fiber-evoked excitatory postsynaptic currents(EPSCs)and inhibitory postsynaptic currents(IPSCs),respectively.Moreover,the Schaffer collateral/commissural pathway was stimulated with paired pulses(interpulse interval was 50 ms)and the paired-pulse facilitation(PPF)was analyzed by EPSC2/EPSC1(P2/P1)ratio.Results PNS(0.1—0.4 g/L)significantly depressed amplitude of EPSCs in neurons in the hippocampal CA1 region(P0.05).Conclusion The inhibitory effect of PNS on EPSCs in hippocampal CA1 pyramidal neurons is not due to the reinforcement of the inhibiting interneurons.It may be a result of direct inhibition on excitatory synaptic transmission.The increasing of P2/P1 ratio after PNS application suggests that PNS depresses the excitatory synaptic transmission by presynaptic mechanism.