Marc L. Turner

Toward the development of a global induced pluripotent stem cell library

The ability to preselect the donor genotype of iPSC lines provides important opportunities for immune matching in cell therapy. Here we propose that an international assessment should be made of how immune incompatibility can best be managed and how a network of GMP HLA homozygous haplobanks could be operated.

Late Outgrowth Endothelial Cells Resemble Mature Endothelial Cells and Are Not Derived from Bone Marrow

A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte‐colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34+CD133−CD146+ cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real‐time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 ± 2.7 vs. 6.6 ± 3.7 vessels, respectively, n = 9). Bone marrow‐derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34+CD133−CD146+ mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrow‐derived circulating precursor for endothelial cells. STEM CELLS2013;31:338–348

Harmonizing standards for producing clinical-grade therapies from pluripotent stem cells

volume 32 NumBeR 8 AuGuST 2014 nature biotechnology conceived for somatic-cell therapies will have to be modified5,6. For example, use of iPSCs may require special guidance with respect to tumorigenicity, genetic integrity, release assays and sterility/aseptic processes. Confusion will arise if existing guidelines are inappropriately adapted or protocols are inadequately generalized to all cell types7–9. It is important to recognize that producing a clinical-grade, PSC-based therapy involves more than complying with cGMP and CMC manufacturing standards. Additional issues that must be considered include regulations on sourcing of donor tissue, ethical guidelines, intellectual-property law and data sharing. Figure 1 outlines the hurdles that arise at different stages of product development. Many of the issues summarized in Figure 1 remain unresolved. For example, in manufacturing, new reference or control material is needed to generate convincing data on in-process testing, lot-to-lot variability and release assays. Guidelines for tissue collection, ownership and payment for PSC generation are in flux10. Equally important, questions regarding consent for the hundreds of thousands of existing samples that could be used as a source of iPSCs must be addressed. More generally, there are uncertainties in how to extend the regulations and standards of institutional review boards, HIPPA (Health Insurance Portability and Privacy Act) and OHRP (Office for Human Research Protection) to PSC-based therapies10,11. This new class of therapy may also require new interpretations of ethical guidelines12, patent law13,14 and the unique propertyrights issues that arise for cells that can make gametes15. To the Editor: Generating clinical-grade cells from pluripotent stem cells (PSCs) for use in patients is not simply a matter of complying with current good manufacturing practices (cGMPs) and chemistry and manufacturing controls (CMCs). A range of other issues demand careful attention, including accessing tissue in an ethical manner and adhering to the varied rules and regulations of specific local and national jurisdictions. The current patchwork of practices represents a major hindrance to progress in regenerative medicine. We propose the establishment of an international body tasked with developing, evaluating and harmonizing the technical, ethical, legal and regulatory frameworks that govern the production of therapies based on PSCs. All PSC-based therapies involve the in vitro conversion of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into differentiated cells that migrate, integrate, survive and function therapeutically in patients1,2. These therapies will be administered by different routes, alone or in combination with biologic or synthetic materials, for a variety of indications, and will require different final-product formulations. Although no PSC-based therapy has yet been approved, at least six or seven groups have commenced or are planning early-stage clinical trials. Unlike somatic cells, PSCs are immortal and have the potential to make any differentiated cell type. These differences have important consequences at all stages of clinical translation, manufacture and commercialization, including requirements for shipping, tracking and identity specifications. Therapies based on somatic cells (including multipotent stem cells or other nonpluripotent cell types) involve collecting cells from a particular donor followed by limited growing, testing, storing and banking of the cells. Manufacture of somatic cell–based therapies involves myriad challenges, including compliance with cGMP and CMC regulations, scale-up and scale-out, and appropriate in-process testing and sterility and potency assays. But therapies based on PSCs bring additional layers of complexity. The cells must undergo extensive expansion and long differentiation procedures to generate appropriate phenotypes while eliminating unwanted phenotypes, including residual pluripotent cells3,4. The use of iPSCs as an autologous product that may be genetically modified raises further issues related to small lot sizes and lack of a master cell bank as in allogeneic therapies. The unique challenges associated with PSC-based therapies are summarized in Table 1. In our view, the manufacturing challenges specific to these therapies mean that existing cGMP and CMC regulations

Identification of the niche and phenotype of the first human hematopoietic stem cells.

Summary In various vertebrate species, the dorsal aorta (Ao) is the site of specification of adult hematopoietic stem cells (HSCs). It has been observed that the upregulation of essential hematopoietic transcription factors and the formation of specific intra-aortic hematopoietic cell clusters occur predominantly in the ventral domain of the Ao (AoV). In the mouse, the first HSCs emerge in the AoV. Here, we demonstrate that in the human embryo the first definitive HSCs also emerge asymmetrically and are localized to the AoV, which thus identifies a functional niche for developing human HSCs. Using magnetic cell separation and xenotransplantations, we show that the first human HSCs are CD34+VE-cadherin+CD45+C-KIT+THY-1+Endoglin+RUNX1+CD38−/loCD45RA−. This population harbors practically all committed hematopoietic progenitors and is underrepresented in the dorsal domain of the Ao (AoD) and urogenital ridges (UGRs). The present study provides a foundation for analysis of molecular mechanisms underpinning embryonic specification of human HSCs.

Granulocyte colony-stimulating factor (G-CSF) depresses angiogenesis in vivo and in vitro: implications for sourcing cells for vascular regeneration therapy

Summary.u2002 Background:u2002The most common source of hematopoietic progenitor cells (HPCs) for hematopoietic reconstitution comprises granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood stem cells (PBSCs). It has been proposed that endothelial progenitor cells (EPCs) share precursors with HPCs, and that EPC release may accompany HPC mobilization to the circulation following G‐CSF administration. Objective:u2002To investigate EPC activity following HPC mobilization, and the direct effects of exogenous G‐CSF administration on human umbilical vein endothelial cells (HUVECs) and endothelial outgrowth cells (EOCs), using in vitro and in vivo correlates of angiogenesis. Patients/Methods:u2002Heparinized venous blood samples were collected from healthy volunteers and from cord blood at parturition. G‐CSF‐mobilized samples were collected before administration, at apheresis harvest, and at follow‐up. PBSCs were phenotyped by flow cytometry, and cultured in standard colony‐forming unit (CFU)‐EPC and EOC assays. The effect of exogenous G‐CSF was investigated by addition of it to HUVECs and EOCs in standard tubule formation and aortic ring assays, and in an in vivo sponge implantation model. Results:u2002Our data show that G‐CSF mobilization of PBSCs produces a profound, reversible depression of circulating CFU‐EPCs. Furthermore, G‐CSF administration did not mobilize CD34+CD133− cells, which include precursors of EOCs. No EOCs were cultured from any mobilized PBSCs studied. Exogenous G‐CSF inhibited CFU‐EPC generation, HUVEC and EOC tubule formation, microvessel outgrowth, and implanted sponge vascularization in mice. Conclusions:u2002G‐CSF administration depresses both endothelial cell angiogenesis and monocyte proangiogenic activity, and we suggest that any angiogenic benefit observed following implantation of cells mobilized by G‐CSF may come only from a paracrine effect from HPCs.

Dissociation of phenotypic and functional endothelial progenitor cells in patients undergoing percutaneous coronary intervention

Objectives: Endothelial progenitor cells (EPCs) are circulating mononuclear cells with the capacity to mature into endothelial cells and contribute to vascular repair. We assessed the effect of local vascular injury during percutaneous coronary intervention (PCI) on circulating EPCs in patients with coronary artery disease. Design and setting: Prospective case-control study in a university teaching hospital. Patients: 54 patients undergoing elective coronary angiography. Interventions and main outcome measures: EPCs were quantified by flow cytometry (CD34+KDR+ phenotype) complemented by real-time polymerase chain reaction (PCR), and the colony forming unit (CFU-EC) functional assay, before and during the first 24 hours after diagnostic angiography (nu200a=u200a27) or PCI (nu200a=u200a27). Results: Coronary intervention, but not diagnostic angiography, resulted in an increase in blood neutrophil count (p<0.001) and C-reactive protein concentrations (pu200a=u200a0.001) in the absence of significant myocardial necrosis. Twenty-four hours after PCI, CFU-ECs increased threefold (median [IQR], 4.4 [1.3–13.8] vs 16.0 [2.1–35.0], pu200a=u200a0.01), although circulating CD34+KDR+ cells (0.019% (SEM 0.004%) vs 0.016% (0.003%) of leucocytes, pu200a=u200a0.62) and leucocyte CD34 mRNA (relative quantity 2.3 (0.5) vs 2.1 (0.4), pu200a=u200a0.21) did not. There was no correlation between CFU-ECs and CD34+KDR+ cells. Conclusions: Local vascular injury following PCI results in a systemic inflammatory response and increases functional CFU-ECs. This increase was not associated with an early mobilisation of CD34+KDR+ cells, suggesting these cells are not the primary source of EPCs involved in the immediate response to vascular injury.

Novel Biopolymers to Enhance Endothelialisation of Intra-vascular Devices

Rapid endothelisation is of critical importance in the prevention of adverse remodelling after device implantation. Currently, there is a need for alternative strategies to promote re-endothelialisation for intravascular stents and vascular grafts. Using polymer microarray technology 345 polymers are comprehensively assessed and a matrix is identified that specifically supports both progenitor and mature endothelial cell activity in vitro and in vivo while minimising platelet attachment.

Human embryonic stem cells rapidly take up and then clear exogenous human and animal prions in vitro.

Susceptibility to prion infection involves interplay between the prion strain and host genetics, but expression of the host‐encoded cellular prion protein is a known prerequisite. Here we consider human embryonic stem cell (hESC) susceptibility by characterizing the genetics and expression of the normal cellular prion protein and by examining their response to acute prion exposure. Seven hESC lines were tested for their prion protein gene codon 129 genotype and this was found to broadly reflect that of the normal population. hESCs expressed prion protein mRNA, but only low levels of prion protein accumulated in self‐renewing populations. Following undirected differentiation, up‐regulation of prion protein expression occurred in each of the major embryonic lineages. Self‐renewing populations of hESCs were challenged with infectious human and animal prions. The exposed cells rapidly and extensively took up this material, but when the infectious source was removed the level and extent of intracellular disease‐associated prion protein fell rapidly. In the absence of a sufficiently sensitive test for prions to screen therapeutic cells, and given the continued use of poorly characterized human and animal bioproducts during hESC derivation and cultivation, the finding that hESCs rapidly take up and process abnormal prion protein is provocative and merits further investigation. Copyright

Red blood cells from pluripotent stem cells for use in transfusion

The use of donated red blood cells in transfusion is a well-established cellular therapy. However, problems including insufficient supply, transfusion-transmitted infections and the need for immunological matching hamper even in the best services. These issues may be eliminated by using pluripotent stem cells to generate universal donor group O, Rhesus D-negative red blood cells. Human embryonic stem cells can be maintained and expanded indefinitely and can, therefore, produce the very large cell numbers required for this application. Red blood cell production is also an attractive goal for pluripotent stem cell-derived therapeutics because it is a well-characterized single cell suspension, lacking nucleated cells and with a low expression of HLA molecules. Much progress has been made; however, a number of challenges remain including scale-up, clinical effectiveness and product safety.

Postmenstrual gestational age should be used with care in studies of early human hematopoietic development

To the editor:nnThe hematopoietic system undergoes rapid changes during embryogenesis; therefore, studying this process requires accurate embryo staging. In the mouse, timed pregnancies can routinely be set and controlled; however, accurate staging of human pregnancies is more problematic,