Mary Kearns-Jonker

Efficient Generation of Integration-Free iPS Cells from Human Adult Peripheral Blood Using BCL-XL Together with Yamanaka Factors

The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source—peripheral blood—has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34+ cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3−/CD19− cells or T/B cell-depleted MNCs for 4–6 days led to the generation of 20–30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

Pig aortic endothelial cell antigens recognized by human IgM natural antibodies.

Human-to-pig xenoantibodies may constitute a major obstacle to the successful use of pigs as xenograft donors for human transplantation. Our studies demonstrate that normal human serum contains antibodies, primarily IgM, that are cytotoxic for pig aortic endothelial cells (PAECs). These antibodies bind to several antigens isolated from PAECs, lymphocytes, platelets, red blood cells, and the kidney. Absorption of human serum with pig lymphocytes removes the cytotoxic activity to PAECs and some, but not all, of the IgM antibodies capable of binding in an ELISA assay to the PAECs. The cytotoxic antibodies are inactivated by 2-mercaptoethanol, suggesting that they are primarily IgM. Whole cell extracts of PAEC, lymphocytes, platelets, red blood cells, and kidney were prepared and analyzed by Western blots to establish the cellular distribution of the xenoantigens that react with human IgM in pooled human serum. Results showed that several of the most intensely stained bands migrated between 24 and 66 kDa. High molecular weight bands (>100 kDa) were observed only in kidney, platelet, and PAEC preparations. Human IgM xeniantibodies also reacted strongly in Western blots to endothelial cell membranes proteins with molecular weights of 62, 48, 42, 36, 34, 28, and 26 kDa. Absorption of human serum with pig lymphocytes removes IgM binding to all bands except for a 34-kDa protein, suggesting this protein is unique to PAECs. Treatment of the PAEC membrane proteins with proteinase K disrupts the binding of the human IgM antibodies. Similar treatment with glycosidases (neuramidase, O-glycosidase, and N-glycosidase F) resulted in a decrease in molecular weight of the 28− and 26-kDa bands, suggesting that these xenoantigens are glycoproteins and that antibody binding to some xenoantigens may not require glycosylation.

Characteristics of immunoglobulin gene usage of the xenoantibody binding to gal-alpha(1,3)gal target antigens in the gal knockout mouse.

BACKGROUND Natural antibodies that react with galactose-alpha(1,3)galactose [galalpha(1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibodies to environmental microbes that express the galalpha(1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-galalpha(1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to this carbohydrate epitope. METHODS Six hybridomas that produce monoclonal antibodies (mAbs) to galalpha(1,3)gal were generated in Gal o/o mice. The mAbs were tested to characterize the binding activity with flow cytometry using pig aortic endothelial cells and ELISA with galalpha(1,3)gal carbohydrates. The VH and VK genes of these hybridomas were cloned, sequenced, and analyzed. RESULTS The mAbs showed distinct patterns of antibody binding to galalpha(1,3)gal antigens. The VH genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configuration. Four of six clones used closely related progeny of the same VH germline gene (VH441). Comparison of the mouse gene VH441 to the human gene IGHV3-11, a gene that encodes antibody activity to galalpha(1,3)gal in humans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (complimentary determining regions 1 and 2) of their immunoglobulin VH genes. CONCLUSIONS These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to galalpha(1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of antibody/antigen interactions associated with the humoral response to pig xenografts in humans.

Xenoantibody response to porcine islet cell transplantation using GTKO, CD55, CD59, and fucosyltransferase multiple transgenic donors.

Promising developments in porcine islet xenotransplantation could resolve the donor pancreas shortage for patients with type 1 diabetes. Using α1,3‐galactosyltransferase gene knockout (GTKO) donor pigs with multiple transgenes should extend xenoislet survival via reducing complement activation, thrombus formation, and the requirement for exogenous immune suppression. Studying the xenoantibody response to GTKO/hCD55/hCD59/hHT islets in the pig‐to‐baboon model, and comparing it with previously analyzed responses, would allow the development of inhibitory reagents capable of targeting conserved idiotypic regions.

The Anti‐Non‐Gal Xenoantibody Response to Xenoantigens on Gal Knockout Pig Cells Is Encoded by a Restricted Number of Germline Progenitors

Antibodies directed at non‐gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into nonhuman primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgVH genes that encode antibody responses to non‐gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3–21 germline progenitor encode xenoantibodies directed at non‐gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine α1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of ‘anti‐non‐gal’ xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti‐non‐gal xenoantibodies cross react with carbohydrate xenoantigens. The galactosyltransferase two gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti‐non‐gal xenoantibodies in primates are encoded by IgVH genes that are restricted to IGHV3–21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.

Xenoantibodies to pig endothelium are expressed in germline configuration and share a conserved immunoglobulin VH gene structure with antibodies to common infectious agents.

BACKGROUND The rejection of pig xenografts in humans is initiated by preformed antibodies that may be related to the natural antibodies that formulate a first line of defense against infectious agents. Immunoglobulin gene variable domains encoding the antibodies that react with similar epitopes expressed on xenoantigens and bacteria may share structurally similar antigen-binding site configurations. METHODS We sequenced the VH immunoglobulin genes and germline progenitors of two rat monoclonal antibodies that recognize pig xenoantigens. Nucleic and amino acid sequences of these xenoantibodies were compared with immunoglobulin genes encoding antibodies that react with bacteria or viruses. RESULTS AND CONCLUSIONS VH genes encoding rat anti-pig xenoantibodies are expressed in germline configuration and share structural similarities, including identical amino acids in key antigenic contact sites that define antibody canonical structural groups, with antibodies to infectious agents.

Isolation, Characterization, and Spatial Distribution of Cardiac Progenitor Cells in the Sheep Heart

BACKGROUND Laboratory large animal models are important for establishing the efficacy of stem cell therapies that may be translated into clinical use. The similarity of ovine and human cardiovascular systems provides an opportunity to use the sheep as a large animal model in which to optimize cell-based treatments for the heart. Recent clinical trials in humans using endogenous cardiovascular progenitor cells report significant improvement in cardiac function following stem cell-based therapy. To date, however, endogenous cardiovascular progenitor cells have not been isolated from the sheep heart. METHODS Cardiovascular cells expressing SSEA-4, CD105 and c-kit were isolated by flow cytometry and cloned from the right atrium of neonatal sheep. The expression of GATA-4, c-kit, and Isl1 was identified by PCR in the cloned cells. Immunohistochemical staining was used to compare the number of SSEA-4 positive cells in the right auricle, right atrium, left ventricle and the apex of the heart of fetal, neonatal and adult sheep. The number of SSEA4+cells was also compared in fetal, pregnant and non-pregnant adult sheep. RESULTS Four distinct cardiac progenitor cell sub-populations were identified in sheep, including CD105+SSEA-4+c-kit+Isl1+GATA-4+cells, CD105+SSEA-4+c-kit+Isl1+GATA-4-cells, CD105+SSEA-4-c-kit-Isl1+GATA-4-cells, and CD105+SSEA-4-c-kit+Isl1+GATA-4-cells. Immunohistochemical staining for SSEA-4 showed that labeled cells were most abundant in the right atrium of fetal hearts where niches of progenitor cells could be identified. CONCLUSION We determined the phenotype and distribution of cardiac progenitor cells in the sheep heart. The availability of cloned endogenous cardiac progenitor cells from sheep will provide a valuable resource for optimizing the conditions for cardiac repair in the ovine model.

Use of molecular modeling and site-directed mutagenesis to define the structural basis for the immune response to carbohydrate xenoantigens

BackgroundNatural antibodies directed at carbohydrates reject porcine xenografts. They are initially expressed in germline configuration and are encoded by a small number of structurally-related germline progenitors. The transplantation of genetically-modified pig organs prevents hyperacute rejection, but delayed graft rejection still occurs, partly due to humoral responses. IgVH genes encoding induced xenoantibodies are predominantly, not exclusively, derived from germline progenitors in the VH3 family. We have previously identified the immunoglobulin heavy chain genes encoding VH3 xenoantibodies in patients and primates. In this manuscript, we complete the structural analysis of induced xenoantibodies by identifying the IgVH genes encoding the small proportion of VH4 xenoantibodies and the germline progenitors encoding xenoantibody light chains. This information has been used to define the xenoantibody/carbohydrate binding site using computer-simulated modeling.ResultsThe VH4-59 gene encodes antibodies in the VH4 family that are induced in human patients mounting active xenoantibody responses. The light chain of xenoantibodies is encoded by DPK5 and HSIGKV134. The structural information obtained by sequencing analysis was used to create computer-simulated models. Key contact sites for xenoantibody/carbohydrate interaction for VH3 family xenoantibodies include amino acids in sites 31, 33, 50, 57, 58 and the CDR3 region of the IgVH gene. Site-directed mutagenesis indicates that mutations in predicted contact sites alter binding to carbohydrate xenoantigens. Computer-simulated modeling suggests that the CDR3 region directly influences binding.ConclusionXenoantibodies induced during early and delayed xenograft responses are predominantly encoded by genes in the VH3 family, with a small proportion encoded by VH4 germline progenitors. This restricted group can be identified by the unique canonical structure of the light chain, heavy chain and CDR3. Computer-simulated models depict this structure with accuracy, as confirmed by site-directed mutagenesis. Computer-simulated drug design using computer-simulated models may now be applied to develop new drugs that may enhance the survival of xenografted organs.

The anti-nongal xenoantibody response to α1,3-galactosyltransferase gene knockout pig xenografts

Purpose of reviewAnti-nonGal xenoantibodies are a major barrier to the survival of genetically modified porcine xenografts. This review summarizes the contribution of anti-nonGal xenoantibodies to the activation of porcine endothelial cells and graft rejection, and further provides an update on recent advancements in defining the unique features of anti-nonGal xenoantibody structure. Recent findingsAnti-nonGal xenoantibodies pre-exist at low levels in humans and nonhuman primates, and are notably absent in neonates. Exposure of nonhuman primates to α1,3-galactosyltransferase gene knockout endothelial cells initiates an induced xenoantibody response that is restricted and encoded by the germline immunoglobulin heavy chain gene IGHV3-21. The target xenoantigen remains undetermined, but several candidate targets have been proposed, including carbohydrate xenoantigens. New advancements in molecular modeling provide insight on the mechanism by which xenoantibodies bind to structurally related carbohydrates. SummaryGenetic manipulation of porcine donors has significantly prolonged the survival of grafts placed into nonhuman primate recipients, but anti-nonGal xenoantibodies and thrombosis limit the ability of these grafts to function on a long-term basis. Recent developments defining pre-existing anti-nonGal xenoantibody levels, the restriction in the anti-nonGal xenoantibody response and the identification of key sites defining xenoantibody–carbohydrate interactions now provide the information necessary to develop new approaches to preventing xenoantibody-mediated rejection.

The humoral response to xenografts is controlled by a restricted repertoire of immunoglobulin VH genes.

BACKGROUND The early phases of the host immune response to xenografts are dominated by anti-donor antibodies. The immunological pathways responsible for mediating the host humoral responses to xenografts are largely unknown, and this report addresses the nature of the immunoglobulin genes controlling the host antibody response to xenografts. METHODS cDNA libraries established from rat anti-hamster monoclonal antibodies and splenic lymphocytes from LEW rats rejecting hamster heart xenografts were used to clone, sequence, and identify the immunoglobulin genes responsible for encoding rat xenoantibodies to hamster heart grafts. Libraries for germline variable region heavy chain (VH) genes encoding the anti-hamster xenograft antibodies were established by genomic DNA cloning and analyzed by nucleotide sequencing. The frequency of Ig VH gene usage for controlling the antibody responses to hamster xenografts was examined by colony-filter dot hybridization. The nucleic acid structure of these genes was then compared to their genomic progenitors to identify the number and structural diversity expressed by the Ig VH genes used to mediate the response. RESULTS Rat monoclonal antibodies selected for their ability to precipitate the rejection of hamster xenografts exclusively use a closely related group of VH genes. The VH genes used by these antibodies are restricted to a single family of germline genes (VHHAR) for which 15 family members have been identified. The frequency of VHHAR gene usage in splenic IgM-producing B cells from LEW rats rapidly expands from 0.8% in naive animals to 13% in recipients 4 days after xenotransplantation. cDNA libraries expressing VHHAR genes were established from splenic lymphocytes derived from naive or xenograft recipients at 4 and 21 days after transplantation. Examination of 20 cDNA clones revealed that the majority (75%) of these clones express VHHAR genes displaying limited somatic mutation. CONCLUSIONS The use of a closely related group of Ig VH genes in a germline configuration to control the early humoral response to xenografts suggests that this response may represent the utilization of a primitive, T cell-independent pathway of antibody production by the graft recipients.