• FVIII
• Hemophilia A
• bone marrow
• cell therapy
• efficacy & safety
• gene therapy
• mesenchymal stroma cell

• FVIII
• endothelial progenitor cell
• gene therapy
• hemophilia A
• mesenchymal stromal (stem) cell therapy

• R01 HL130856 NHLBI NIH HHS
• R01 HL135853 NHLBI NIH HHS
• U01 HL148681 NHLBI NIH HHS
• UL1 TR001420 NCATS NIH HHS

• Animals
• Female
• Genetic Diseases, Inborn/therapy
• Genetic Therapy/methods
• Humans
• Pregnancy
• Prenatal Care/methods
• Stem Cell Transplantation/methods

• R01 HL135853 NHLBI NIH HHS
• R01 HL146128 NHLBI NIH HHS
• U01 HL148681 NHLBI NIH HHS

The delivery of factor VIII (FVIII) through gene and/or cellular platforms has emerged as a promising hemophilia A treatment. Herein, we investigated the suitability of human placental cells (PLCs) as delivery vehicles for FVIII and determined an optimal FVIII transgene to produce/secrete therapeutic FVIII levels from these cells. Using three PLC cell banks we demonstrated that PLCs constitutively secreted low levels of FVIII, suggesting their suitability as a transgenic FVIII production platform. Furthermore, PLCs significantly increased FVIII secretion after transduction with a lentiviral vector (LV) encoding a myeloid codon-optimized bioengineered FVIII containing high-expression elements from porcine FVIII. Importantly, transduced PLCs did not upregulate cellular stress or innate immunity molecules, demonstrating that after transduction and FVIII production/secretion, PLCs retained low immunogenicity and cell stress. When LV encoding five different bioengineered FVIII transgenes were compared for transduction efficiency, FVIII production, and secretion, data showed that PLCs transduced with LV encoding hybrid human/porcine FVIII transgenes secreted substantially higher levels of FVIII than did LV encoding B domain-deleted human FVIII. In addition, data showed that in PLCs, myeloid codon optimization is needed to increase FVIII secretion to therapeutic levels. These studies have identified an optimal combination of FVIII transgene and cell source to achieve clinically meaningful levels of secreted FVIII.

• ET3
• FVIII
• HSQ
• cell therapy
• codon-optimization
• gene therapy
• hemophilia A
• placental cells

Mesenchymal stromal cells (MSCs) possess several fairly unique properties that, when combined, make them ideally suited for cellular-based immunotherapy and as vehicles for gene and drug delivery for a wide range of diseases and disorders. Key among these are: (1) their relative ease of isolation from a variety of tissues; (2) the ability to be expanded in culture without a loss of functionality, a property that varies to some degree with tissue source; (3) they are relatively immune-inert, perhaps obviating the need for precise donor/recipient matching; (4) they possess potent immunomodulatory functions that can be tailored by so-called licensing in vitro and in vivo; (5) the efficiency with which they can be modified with viral-based vectors; and (6) their almost uncanny ability to selectively home to damaged tissues, tumors, and metastases following systemic administration. In this review, we summarize the latest research in the immunological properties of MSCs, their use as immunomodulatory/anti-inflammatory agents, methods for licensing MSCs to customize their immunological profile, and their use as vehicles for transferring both therapeutic genes in genetic disease and drugs and genes designed to destroy tumor cells.

• Mesenchymal stem cells
• Stem cells
• Type 1 diabetes

• biological bystander effects
• human hematopoiesis
• space radiation

• Film and medicine
• Ivan Illich
• expropriation of death
• ontology
• screen
• xenotransplantation

Recent advances in high-throughput molecular testing have made it possible to diagnose most genetic disorders relatively early in gestation with minimal risk to the fetus. These advances should soon allow widespread prenatal screening for the majority of human genetic diseases, opening the door to the possibility of treatment/correction prior to birth. In addition to the obvious psychological and financial benefits of curing a disease in utero, and thereby enabling the birth of a healthy infant, there are multiple biological advantages unique to fetal development, which provide compelling rationale for performing potentially curative treatments, such as stem cell transplantation or gene therapy, prior to birth. Herein, we briefly review the fields of in utero transplantation (IUTx) and in utero gene therapy and discuss the biological hurdles that have thus far restricted success of IUTx to patients with immunodeficiencies. We then highlight several recent experimental breakthroughs in immunology, hematopoietic/marrow ontogeny, and in utero cell delivery, which have collectively provided means of overcoming these barriers, thus setting the stage for clinical application of these highly promising therapies in the near future.

• Alternate sources of HSC
• Bone marrow
• HSC for repairing non-hematopoietic tissues
• HSC for tolerance induction
• Hematopoietic stem cell (HSC)
• Transplantation

• fetal intervention
• hemophilia
• immune tolerance
• in utero gene therapy
• in utero transplantation
• mesenchymal stromal cells
• sheep model

• R01 HD043038 NICHD NIH HHS
• R01 HL073737 NHLBI NIH HHS
• R01 HL135853 NHLBI NIH HHS
• U01 HL148681 NHLBI NIH HHS

• HPCs niche
• differentiation
• hepatic progenitor cells (HPCs)
• liver regeneration

• Bone Marrow Cells/physiology
• Cell Differentiation/physiology
• Cell- and Tissue-Based Therapy/methods
• Cell- and Tissue-Based Therapy/trends
• Hepatocytes/cytology
• Humans
• Immunophenotyping
• Liver Regeneration/physiology
• Stem Cells/immunology
• Stem Cells/physiology

• Cytomegalovirus
• HCMV
• hematopoietic niche
• pericytes
• transplantation

• FVIII
• Gene therapy
• Hemophilia A
• MSCs
• Transgenic mouse

In utero stem cell transplantation, which promises treatment for a host of genetic disorders early in gestation before disease effect stems from Ray Owen’s seminal observation that self-tolerance, is acquired during gestation. To date, in utero transplantation (IUT) has proved useful in characterizing the hematopoietic stem cell. Recent observations support its use as an in vivo method to further understanding of self-tolerance. Preclinical development continues for its application as a treatment for childhood hematolymphoid diseases. In addition, IUT may offer therapeutic options in the treatment of diabetes among other diseases. Thus IUT serves as a technique or system important in both a basic and applied format. This review summarizes these findings.

• Self non-self discrimination
• Immune ontogeny
• Stem cell transplantation

Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express molecules, such as membrane cofactor protein (CD46), complement decay-accelerating factor (CD55), and protectin (CD59), which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. We have previously demonstrated that transduction of MSC with a retrovirus encoding HCMV-US proteins resulted in higher levels of MSC engraftment due to decreased HLA-I expression. Here, we investigate whether engineering MSC to express US2 (MSC-US2), US3 (MSC-US3), US6 (MSC-US6), or US11 (MSC-US11) HCMV proteins can alter complement recognition, thereby better protecting MSC from complement attack and lysis. HCMV-US proteins increased MSC CD59 expression at different levels as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI). A significant increase in CD59 expression was seen in MSC-US2, MSC-US3, and MSC-US6, but not in MSC-US11. Only MSC-US2 displayed increased expression of CD46, while US2 and US3 proteins were both able to augment the percentage of MSC expressing this molecule. Regardless of the HCMV protein expressed, none changed CD55 MFI; however, expression of US6, US11, and US2 each increased the percentage of MSC that were positive for this molecule. Because US2 protein was the most efficient in up-regulating all three complement regulatory proteins, we used a functional complement-mediated cytotoxicity assay to investigate whether MSC-US2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis by 59.10±12.89% when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV-US proteins in complement evasion, and our data shows that over-expression of US2 protein on MSC could serve as a strategy to protect these cells from complement lysis.

• Lgr5
• Musashi-1
• crypts of Lieberkühn
• epithelial
• niche
• transcriptome
• transplant

• intestinal crypt
• vascular bed
• cd133
• c-kit
• myofibroblasts

Stem cell therapy hold the potential to meet the demand for transplant cells/tissues needed for treating damages resulting from both natural and man-made disasters. Pluripotency makes embryonic stem cells and induced pluripotent stem cells ideal for use, but their teratogenic character is a major hindrance. Therapeutic benefits of bone marrow transplantation are well known but characterizing the potentialities of haematopoietic and mesenchymal cells is essential. Haematopoietic stem cells (HSCs) have been used for treating both haematopoietic and non-haematopoietic disorders. Ease of isolation, in vitro expansion, and hypoimmunogenecity have brought mesenchymal stem cells (MSCs) into limelight. Though differentiation of MSCs into tissue-specific cells has been reported, differentiation-independent mechanisms seem to play a more significant role in tissue repair which need to be addressed further. The safety and feasibility of MSCs have been demonstrated in clinical trials, and their use in combination with HSC for radiation injury treatment seems to have extended benefit. Therefore, using stem cells for treatment of disaster injuries along with the conventional medical practice would likely accelerate the repair process and improve the quality of life of the victim.

Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity.

We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses.

These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch.

• hematopoietic stem cells
• mesenchymal stem cells
• hepatocytes
• liver regeneration
• cell therapy

• Bone Marrow Cells/cytology
• Humans
• Liver Diseases/therapy
• Liver Regeneration
• Stem Cell Transplantation
• Stem Cells/cytology

• R01 HL070566 NHLBI NIH HHS
• HL97623 NHLBI NIH HHS
• HL52955 NHLBI NIH HHS
• R01 HL052955 NHLBI NIH HHS
• HL73737 NHLBI NIH HHS
• R01 HL073737 NHLBI NIH HHS
• R01 HL097623 NHLBI NIH HHS

• pancreatic mesenchymal stem cells
• in utero stem cell transplantation
• type 1 diabetes

• Animals
• Cell Differentiation
• Cells, Cultured
• Female
• Flow Cytometry
• Humans
• Immunohistochemistry
• Insulin/blood
• Insulin/metabolism
• Islets of Langerhans/metabolism
• Mesenchymal Stem Cell Transplantation
• Mesenchymal Stem Cells/cytology
• Pregnancy
• Reference Standards
• Reverse Transcriptase Polymerase Chain Reaction
• Sheep

• R01 HL052955 NHLBI NIH HHS
• R37 HL049042 NHLBI NIH HHS
• HL49042 NHLBI NIH HHS
• HL52955 NHLBI NIH HHS