Engraftment Of Pluripotent Stem Cells Research Articles

Pluripotent stem cells and tolerance induction in organ transplantation.

Ongoing research is constantly looking for means to modulate the immune system for long-lasting engraftment of pluripotent stem cells (PSC) during stem cell-based therapies. This study reviews data on in-vitro and in-vivo immunogenicity of embryonic and induced-PSC and describes how their immunological properties can be harnessed for tolerance induction in organ transplantation. Although PSC display immunomodulatory properties in vitro, they are capable of eliciting an immune response that leads to cell rejection when transplanted into immune-competent recipients. Nevertheless, long-term acceptance of PSC-derived cells/tissues in an allogeneic environment can be achieved using minimal host conditioning. Protocols for differentiating PSC towards haematopoietic stem cells, thymic epithelial precursors, dendritic cells, regulatory T cells and myeloid-derived suppressor cells are being developed, suggesting the possibility to use PSC-derived immunomodulatory cells to induce tolerance to a solid organ transplant. PSC and/or their derivatives possess unique immunological properties that allow for acceptance of PSC-derived tissue with minimal host conditioning. Investigators involved either in regenerative or in transplant medicine must join their efforts with the ultimate aim of using PSC as a source of donor-specific cells that would create a protolerogenic environment to achieve tolerance in solid organ transplantation.

Professor Dame Julia Polak DBE

Dame Julia Polak, who has died at age 75, pioneered work on histochemistry and then on tissue engineering of the lung. She developed idiopathic pulmonary arterial hypertension, underwent lung transplantation in 1995, and was one of the world's longest-living lung transplant survivors. We remember Julia as a charismatic, inspiring colleague and teacher, strong and feisty but with a very courageous, kind, and gentle heart. Julia Margaret Polak was born on June 26, 1939, in Buenos Aires, Argentina, where her Jewish grandparents had emigrated from Eastern Europe. Her father was a lawyer and her mother a writer. She studied medicine at the University of Buenos Aires. There she met her future husband, Daniel Catovsky, while they were dissecting a corpse. Julia qualified in 1961 and decided to specialize in pathology. Their first child was born in 1963, and the family came to London in 1967, when Daniel was awarded a traveling scholarship. Daniel eventually became an eminent professor of hematology and a world authority on chronic adult leukemias. Julia did her postgraduate studies at the Royal Postgraduate Medical School, Hammersmith Hospital (now part of Imperial College London), and eventually became head of the Histochemistry Department, then Professor of Endocrine Pathology. Julia initially focused on the localization of then-novel peptides, discovered the existence of a hormone system in the gut, and showed that the nervous system is regulated by peptides. Aware of the close relationship between the gut and the lung, Julia began studying the lung in the mid-1980s. She began by localizing various peptides during normal development and in disease and then worked on the nitric oxide pathway. Julia built a strong, very productive research team. Her laboratory meetings were legendary. The team would assemble around the perimeter of her very large office at Hammersmith Hospital, and Julia would ask for updates one by one, senior and junior members alike. Heaven help the researcher who had nothing to report. You knew when she was impressed because her face would light up, and she would say “Fantastic!” wonderfully drawn out and congratulatory. Julia was enjoying a brilliant career when she realized that she was unwell. Initially thought to have asthma, she was in extremis when diagnosed with idiopathic pulmonary arterial hypertension. Professor Sir Magdi Yacoub performed her heart and lung transplant. Julia took the best possible care of her transplant and continued to live life to the full. Memorably, she presented her own case, including the histology of her old lungs, at one of the formidable Hammersmith Grand Rounds. She described in detail the plexiform lesions and vascular pathology. Energized by her own experience, Julia went on to establish the Imperial College Tissue Engineering and Regenerative Medicine Centre and became its professor in 1997. The declared aim of her research was the restoration of normal lung function by the delivery and engraftment of pluripotent stem cells, both exogenous and mobilized endogenous cells, and the creation of a 3-dimensional lung structure that could be implanted into the thorax. These studies necessitated investigation of biomaterials, configured in 3 dimensions with nanomodified surfaces for cell recruitment and differentiation. Dame Julia served on several national and international tissue engineering and stem cell advisory panels. Julia Polak published almost a thousand original papers and made a huge contribution to medical science. She was one of the most highly cited authors of her generation. She received many honors and awards in the United Kingdom and other countries and was appointed DBE (Dame Commander of the Most Excellent Order of the British Empire) by the Queen in 2003. Julia and Daniel Catovsky had 3 children, Marina, Sebastian, and Michael. Marina was tragically killed by a motorcyclist in 2011. Professor Dame Julia Polak DBE was an inspiration to all who knew her. We extend our heartfelt sympathy to her family. Professor Dame Julia Margaret Polak: born June 26, 1939; died August 11, 2014. E-mail: s.haworth@ich.ucl.ac.uk

Reduced Collagen Deposition in Infarcted Myocardium Facilitates Induced Pluripotent Stem Cell Engraftment and Angiomyogenesis for Improvement of Left Ventricular Function

The purpose of this study was to assess the effect of scar tissue composition on engraftment of progenitor cells into infarcted myocardium. Scar tissue formation after myocardial infarction creates a barrier that severely compromises tissue regeneration, limiting potential functional recovery. In vitro: A tricell patch (Tri-P) was created from peritoneum seeded and cultured with induced pluripotent stem cell-derived cardiomyocytes, endothelial cells, and mouse embryonic fibroblasts. The expression of fibrosis-related molecules from mouse embryonic fibroblasts and infarcted heart was measured by Western blot and quantitative reverse transcriptase polymerase chain reaction. In vivo: A Tri-P was affixed over the entire infarcted area 7 days after myocardial infarction in mice overexpressing adenylyl cyclase 6 (AC6). Engraftment efficiency of progenitor cells in hearts of AC6 mice was compared with that of control wild-type (WT) mice using a combination of in vivo bioluminescence imaging, post-mortem ex vivo tissue analysis, and the number of green fluorescent protein-positive cells. Echocardiography of left ventricular (LV) function was performed weekly. Hearts were harvested for analysis 4 weeks after Tri-P application. Mouse embryonic fibroblasts were stimulated with forskolin before an anoxia/reoxygenation protocol. Fibrosis-related molecules were analyzed. In AC6 mice, infarcted hearts treated with Tri-P showed significantly higher bioluminescence imaging intensity and numbers of green fluorescent protein-positive cells than in WT mice. LV function improved progressively in AC6 mice from weeks 2 to 4 and was associated with reduced LV fibrosis. Application of a Tri-P in AC6 mice resulted in significantly higher induced pluripotent stem cell engraftment accompanied by angiomyogenesis in the infarcted area and improvement in LV function.

Role of Blood- and Tissue-Associated Inducible Nitric-Oxide Synthase in Colonic Inflammation

There is evidence that inducible nitric-oxide synthase (iNOS)-derived NO contributes to the pathophysiology of intestinal inflammation. The aims of this study were to assess the role of iNOS in the development of dextran sodium sulfate (DSS)-induced colonic inflammation and to define the contribution of tissue-specific iNOS expression to this inflammatory response. Study groups included: 1) wild-type (WT) mice; 2) WT=>WT bone marrow chimeras with normal iNOS function; 3) WT=>iNOS-/- chimeras (with functional blood cell iNOS, but iNOS-deficient tissue); 4) iNOS-/-=>WT chimeras (with iNOS-deficient blood cells, but normal tissue iNOS activity); and 5) iNOS-deficient mice. In WT mice and WT=>WT chimeras, DSS-induced colonic inflammation was characterized by bloody diarrhea and a high disease activity index. However, WT=>iNOS-/- and iNOS-/-=>WT chimeras and iNOS-/- mice exhibited an attenuated disease activity index, with parallel changes in histopathology. Colonic myeloperoxidase (MPO) was comparably elevated in DSS-treated WT mice (30.1+/-1.7) and WT=>WT chimeras (29.0+/-1), whereas MPO was significantly reduced in iNOS-/- mice and iNOS-/-=>WT chimeras (9.5+/-1.7 and 15.6+/-2.2, respectively). WT=>iNOS-/- chimeras exhibited the lowest MPO activity (3.7+/-0.6). Our findings implicate both blood cell- and tissue-derived iNOS in DSS-induced colonic inflammation, with tissue-associated iNOS making a larger contribution to the recruitment of inflammatory cells.

TRANSPLANTATION OF THE BONE MARROW MICROENVIRONMENT LEADS TO HEMATOPOIETIC CHIMERISM WITHOUT CYTOREDUCTIVE CONDITIONING1

It has been hypothesized that regimens to induce transplantation tolerance and long-term hematopoietic chimerism require recipient conditioning with whole body irradiation or a cytoablative regimen to create space within the marrow microenvironment to permit pluripotent stem cell engraftment. The purpose of this study was to determine if transplantation of an intact bone marrow microenvironment in the form of a bone graft would permit stable hematopoietic stem cell engraftment, shape the repertoire of developing T cells, and induce donor-specific unresponsiveness in the absence of a conditioning regimen. Fragments of femur were transplanted under the kidney capsule of recipient mice. At defined time points after bone graft transplantation recipients were assayed for chimerism, bone graft viability, and responses to donor and third party alloantigens in vitro and in vivo. In the absence of an immunological barrier, bone graft transplantation resulted in long-term multi-lineage hematopoietic chimerism in the peripheral blood. Nude bone graft transplantation into SCID recipients resulted in development of donor- derived T cells that underwent negative selection on bone graft derived I-E+ cells within the thymus. Across a fully allogeneic barrier in immunocompetent recipients treated with combined blockade of the CD40 and CD28 pathways bone graft transplantation resulted in long-term donor-specific hyporesponsiveness in vitro and acceptance of donor specific skin grafts. Transplantation of bone marrow in the form of a bone graft may facilitate the production of hematopoietic chimerism and lead to long-term donor-specific hyporesponsiveness in the absence of a cytoreductive conditioning regimen.

Inability of Cyclophosphamide-Induced Tolerance to Permit Engraftment of Pluripotent Stem Cells Contained in a Moderate Number of Syngeneic Bone Marrow Cells

We have previously described cyclophosphamide (CP)-induced tolerance that comprises an intravenous (i.v.) injection of 1 x 10 8 allogeneic spleen cells (SC) followed, 2 days later, by an intraperitoneal (i.p.) administration of 200 mg/kg of CP. By using this method, we were able to readily induce both long-lasting mixed chimerism and skin allograft tolerance in most of H- 2 matched combinations, but not in H-2 mismatched combinations. In the present study, we have investigated whether the treatment with SC followed by 200 mg/kg CP can permit engraftment of syngeneic pluripotent hematopoietic stem cells (HSC), and whether CP itself can permit the HSC engraftment. Recipient B6 (H-2 b; Ly-5.2) mice received 1 x 10 7, 5 x 10 7 or 1 x 10 8 SC from syngeneic B6.Ly-5.1 (H-2 b; Ly-5.1) mice and administered 2 day later 200 mg/kg CP. Using anti-Ly-5.1 and Ly-5.2 mAbs and a flow cytometry, the origins of lymphoid and myeloid cells injecting the recipients were observed over time. Chimerism was at least initially detectable in all groups treated with SC alone (without 200 mg/kg CP), but became undetectable by 32 weeks after the treatment with SC alone. In recipient B6 mice treated with B6.Ly-5.1 SC and 200 mg/kg CP, on the other hand, lymphoid-chimerism was detectable at 32 weeks in a transferred cell dose-dependent manner, but granulocytechimerism indicating pluripotent HSC engraftment disappeared earlier than 32 weeks after the treatment. In order to further evaluate the effect of CP itself on HSC engraftment, recipient B6 mice were given various doses of CP (50-400 mg/kg) and were injected with T celldepleted 1 × 10 7 BMC from B6.Ly-5.1 mice. Multilineage mixed chimerism over 32 weeks was induced in only one of 11 B6 mice treated with 400 mg/kg CP followed by B6.Ly-5.1 BMC, although lymphoid chimerism was induced temporarily in recipient B6 mice treated with 200 mg/kg CP followed by B6.Ly-5.1 BMC and persistently in most of recipient B6 mice treated with 400 mg/kg CP followed by B6.Ly-5.1 BMC.

Cyclophosphamide 200 mg/kg lacks ability to induce pluripotent stem cell engraftment in mice

Cyclophosphamide 200 mg/kg lacks ability to induce pluripotent stem cell engraftment in mice

Lack of pluripotent stem cell engraftment in cyclophosphamide-induced tolerance

Lack of pluripotent stem cell engraftment in cyclophosphamide-induced tolerance

Myelosuppressive Conditioning Is Required to Achieve Engraftment of Pluripotent Stem Cells Contained in Moderate Doses of Syngeneic Bone Marrow

We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.

Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.