Dachun Wang

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute ≈60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, α-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

Both Cell Fusion and Transdifferentiation Account for the Transformation of Human Peripheral Blood CD34-Positive Cells Into Cardiomyocytes In Vivo

Background—Adult human peripheral blood CD34-positive (CD34+) cells appear to transform into cardiomyocytes in the injured hearts of severe combined immunodeficient mice. It remains unclear, however, whether the apparent transformation is the result of transdifferentiation of the donor stem cells or of fusion of the donor cell with the cardiomyocyte of the recipients. Methods and Results—We performed flow cytometry analyses of cells isolated from the hearts of mice that received human CD34+ cells. Human HLA-ABC antigen and cardiac troponin T or Nkx2.5 were used as markers for cardiomyocytes derived from human CD34+ cells, and HLA-ABC and VE-cadherin were used to identify the transformed endothelial cells. The double-positive cells were collected and interphase fluorescence in situ hybridization was used to detect the expression of human and mouse X chromosomes in these cells. We found that 73.3% of nuclei derived from HLA+ and troponin T+ or Nkx2.5+ cardiomyocytes contain both human and mouse X chromosomes and 23.7% contain only human X chromosome. In contrast, the nuclei of HLA−, troponin T+ cells contain only mouse X chromosomes. Furthermore, 97.3% of endothelial cells derived from CD34+ cells contained human X chromosome only. Conclusions—Thus, both cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo.

SENP1 Enhances Androgen Receptor-Dependent Transcription through Desumoylation of Histone Deacetylase 1

ABSTRACT SUMO (also called Sentrin) is a ubiquitin-like protein that plays an important role in regulating protein function and localization. It is known that several nuclear receptors are modified by SUMO; however, the effect of desumoylation in regulating nuclear receptor function has not been elucidated. Here we show that androgen receptor (AR)-mediated transcription is markedly enhanced by SENP1, a member of SUMO-specific protease family. SENP1s ability to enhance AR-dependent transcription is not mediated through desumoylation of AR, but rather through its ability to deconjugate histone deacetylase 1 (HDAC1), thereby reducing its deacetylase activity. HDAC1s repressive effect on AR-dependent transcription could be reversed by SENP1 and by deletion of its sumoylation sites. RNA interference depletion of endogenous HDAC1 also reduced SENP1s effect. Thus, SENP1 could regulate AR-dependent transcription through desumoylation of HDAC1. These studies provide insights on the potential role of desumoylation in the regulation of nuclear receptor activity.

Transplantation of Human Embryonic Stem Cell–Derived Alveolar Epithelial Type II Cells Abrogates Acute Lung Injury in Mice

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

Targeted Disruption of the Gene Encoding the Murine Small Subunit of Carboxypeptidase N (CPN1) Causes Susceptibility to C5a Anaphylatoxin-Mediated Shock

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits (CPN1) and two large subunits (CPN2) that protect the protein from degradation. Historically, CPN has been implicated as a major regulator of inflammation by its enzymatic cleavage of functionally important arginine and lysine amino acids from potent phlogistic molecules, such as the complement anaphylatoxins C3a and C5a. Because of no known complete CPN deficiencies, the biological impact of CPN in vivo has been difficult to evaluate. Here, we report the generation of a mouse with complete CPN deficiency by targeted disruption of the CPN1 gene. CPN1−/− mice were hypersensitive to lethal anaphylactic shock due to acute complement activation by cobra venom factor. This hypersensitivity was completely resolved in CPN1−/−/C5aR−/− but not in CPN1−/−/C3aR−/− mice. Moreover, CPN1−/− mice given C5a i.v., but not C3a, experienced 100% mortality. This C5a-induced mortality was reduced to 20% when CPN1−/− mice were treated with an antihistamine before C5a challenge. These studies describe for the first time a complete deficiency of CPN and demonstrate 1) that CPN plays a requisite role in regulating the lethal effects of anaphylatoxin-mediated shock, 2) that these lethal effects are mediated predominantly by C5a-induced histamine release, and 3) that C3a does not contribute significantly to shock following acute complement activation.

Characterization of the vls antigenic variation loci of the Lyme disease spirochaetes Borrelia garinii Ip90 and Borrelia afzelii ACAI.

The vls locus of Borrelia burgdorferi B31 consists of 15 silent cassettes and one expression site (vlsE), and the presence of the encoding plasmid lp28‐1 correlates with high infectivity. Recombination between the expression cassette and silent cassettes occurs in vivo, and this process may enable B. burgdorferi to evade the immune response. To determine the characteristics of the vls loci in other Borrelia strains, we have cloned and characterized the vls silent cassette loci of Borrelia garinii Ip90 and Borrelia afzelii ACAI, consisting of 11 vls silent cassettes and 14 vls silent cassettes respectively. The silent cassettes share 90–97% nucleotide sequence identity with one another within the Ip90 vls locus and 84–91% within the ACAI vls locus. In both organisms, the silent cassettes resemble the B31 Vls sequences in overall amino acid similarity (50–65%) and in the presence of six variable regions interspersed between six relatively invariant regions. The vlsE expression sites of these two strains have not been isolated, but transcripts of vlsE were detected by reverse transcriptase‐polymerase chain reaction for both Ip90 and ACAI. In addition, the occurrence of sequence variation within the vlsE cassette region of these transcripts was verified. This study indicates that the vls loci present in B. garinii Ip90 and B. afzelii ACAI have characteristics similar to those found in B. burgdorferi B31.

Therapeutic Potential of Lung Epithelial Progenitor Cells Derived from Embryonic and Induced Pluripotent Stem Cells

Embryonic stem (ES) cells derived from preimplantation blastocysts and induced pluripotent stem (iPS) cells generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide a possible unlimited source of cells that could be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. Because of inherent difficulties in deriving endodermal cells from undifferentiated cell cultures, applications using lung epithelial cells derived from ES and iPS cells have lagged behind similar efforts devoted to other tissues, such as the heart and spinal cord. However, during the past several years, significant advances in culture, differentiation, and purification protocols, as well as in bioengineering methodologies, have fueled enthusiasm for the development of stem cell-based lung therapeutics. This article provides an overview of recent research achievements and discusses future technical challenges that must be met before the promise of stem cell applications for lung disease can be realized.

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA‐I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA‐I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2‐microglobulin on both gene alleles in hESCs. Because β2‐microglobulin serves as the HLA‐I light chain, disruption of the β2‐microglobulin gene led to complete HLA‐I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T‐cell‐mediated destruction. Although interferon‐γ (IFN‐γ) treatment significantly induced β2‐microglobulin expression, promoting CD8+ T cell‐mediated killing of control hESCs and their derivatives, CD8+ T‐cell‐mediated cytotoxicity was barely observed with β2‐microglobulin‐null hESCs and their derivatives treated with IFN‐γ. This genetic manipulation to disrupt HLA‐I expression did not affect the self‐renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell‐mediated killing due to the lack of HLA‐I expression, when transplanted into NK cell‐depleted immunocompetent mice, β2‐microglobulin‐null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2‐microglobulin‐null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future.

Cytokines Increase Neonatal Cardiac Myocyte Calcium Concentrations: The Involvement of Nitric Oxide and Cyclic Nucleotides

Neonatal rat cardiac myocytes were treated with cytokines, with or without the nitric oxide synthase (NOS) inhibitors N-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine methyl ester (LNAME), and systolic and diastolic calcium levels were measured by fluorescence spectrophotometry and confocal microscopy. Time-dependent changes following interferon-gamma (IFN-gamma) treatment revealed a continuing increase in intracellular calcium, which was reduced with LNMMA, but not with LNAME. Increases in calcium also occurred with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not to the extent seen with IFN-gamma. Increased cyclic guanosine monophosphate (cGMP) was involved in the results described with short-term (2 hr) TNF-alpha and long-term (18 hr) IFN-gamma treatments. Short-term exposure to IFN-gamma produced an increase in cyclic adenosine monophosphate (cAMP) and also an initial increase in the myocyte-bearing rate, with calcium levels either (i) subsequently returning to control levels while maintaining a fast beating rate or (ii), retaining a high systolic calcium level, but beating at control rates. Treatment with both IL-1beta and IFN-gamma stabilized the beating rate of the cells on some occasions. Shortening of myocytes increased with isoproterenol and following treatment with IFN-gamma, while isoproterenol stimulation of IFN-gamma-treated cells revealed increased contractile activity after short, but not long, treatment. LNMMA, but not reduced the increased contractile response with short-term IFN-gamma treatment. Our findings suggest that TNF-alpha acts via a cGMP-dependent pathway, whereas the actions of IFN-gamma involve adenylate cyclase, and possibly a NO-forming mechanism and cGMP pathway as well. It is also apparent that the two NO inhibitors function via different mechanisms or that LNMMA has a direct effect on the calcium-signaling pathway.

A Site‐Specific Genetic Modification for Induction of Pluripotency and Subsequent Isolation of Derived Lung Alveolar Epithelial Type II Cells

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC‐derived cell types are still major obstacles. Here we report a novel strategy using a single nonviral site‐specific targeting vector with a combination of Tet‐On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)‐specific NeomycinR transgene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of β‐2‐microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc, and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random integration‐free or genetic mutation‐free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random integration‐free and exogenous reprogramming factor‐free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC‐derived ATIICs exhibit ultrastructural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin‐challenged mice lungs, hiPSC‐derived ATIICs efficiently remain and re‐epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient‐specific ATIICs for possible future clinical applications. Stem Cells 2014;32:402–413