技术资料

Teratoma formation,the standard in vivo pluripotency assay,is also frequently used as a tumorigenicity assay. A common concern in therapeutic stem cell applications is the tumorigenicity potential of a small number of cell impurities in the final product. Estimation of this small number is hampered by the inaccurate methodology of the tumorigenicity assay. Hence,a protocol for tumorigenicity assay that can deliver a defined number of cells,without error introduced by leakage or migration of cells is needed. In this study,we tested our modified transplantation method that allows for transplant of small numbers of pluripotent stem cells (PSCs) under the kidney capsule with minimal cell leakage. A glass capillary with a finely shaped tip and an attached mouth pipette was used to inject PSCs into the rodent kidney capsule. H9 embryonic and induced PSCs were tagged with Fluc and green fluorescence protein reporter genes and divided in different cell doses for transplantation. Bioluminescence imaging (BLI) on the day of surgery showed that the cell signal was confined to the kidney and signal intensity correlated with increasing transplant cell numbers. The overall cell leakage rate was 17% and the rodent survival rate was 96%. Teratoma formation was observed in rodents transplanted with cell numbers between 1 × 10(5)-2 × 10(6). We conclude that this modified procedure for transplanting PSCs under the kidney capsule allows for transplantation of a defined number of PSCs with significant reduction of error associated with cell leakage from the transplant site. View Publication

Hematopoiesis depends on the association of hematopoietic stem cells with stromal cells that constitute the hematopoietic microenvironment. The in vitro development of the endothelial cell from umbilical cord blood (UCB) is not well established and has met very limited success. In this study,UCB CD34(+) cells were cultured for 5 weeks in a stroma-free liquid culture system using thrombopoietin,flt3 ligand,and granulocyte-colony stimulating factor. By week 4-5,we found that firmly adherent fibroblast-like cells were established. These cells showed characteristics of endothelial cells expressing von Willebrand factor,human vascular cell adhesion molecule-1,human intracellular adhesion molecule-1,human CD31,E-selectin,and human macrophage. Furthermore,when comparing an ex vivo system without an established endothelial monolayer to an ex vivo system with an established endothelial monolayer,better expansion of total nucleated cells,CD34(+) cells,and colony-forming units (CFUs)-granulocyte-macrophage and CFUs-granulocyte-erythroid-megakaryocyte-macrophage were found during culture. This phenomenon was in part due to the fact that a significant reduction of apoptotic fractions was found in the CD34(+) cells,which were cultured on the adherent monolayer for up to 5 weeks. To gather quantitative data on the number of endothelial cells derived from a given number of CD34 cells,we performed limiting dilution assay by using Poisson distribution: the number of tested cells (linear scale) producing a 37% negative culture (logarithmic scale) is the number of cells containing one endothelial cell. By this method,one endothelial cell may be found from 314 CD34(+) cells after 5 weeks of culture. These results suggest that the UCB CD34(+) cell fraction contains endothelial cell precursors,establishing the hematopoietic microenvironment and providing the beneficial effects through downregulating apoptosis on UCB expansion protocols. These observations may provide insight for future cellular therapy or graft engineering. View Publication

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH),hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon,we investigated the usefulness of ALDH activity for characterizing HSC graft quality,particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time,and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly,in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells,this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism,whereas all other patients showed early complete donor chimerism. In conclusion,we suggest to measure ALDH activity as a surrogate marker for HSC activity,and to transport and store PBSC under controlled cooling conditions. View Publication

Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes,for pharmacologic studies and as a potential resource for therapeutic transplant. To date,limited in vivo models exist for human intestine,all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here,we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme,both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme,as demonstrated by differentiated intestinal cell lineages (enterocytes,goblet cells,Paneth cells,tuft cells and enteroendocrine cells),presence of functional brush-border enzymes (lactase,sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore,transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection,suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology,disease and translational studies. View Publication

The CD45 antigen is present on all cells of the hematopoietic lineage. Using a murine model,we have determined whether a lytic CD45 monoclonal antibody can produce persistent aplasia and whether it could facilitate syngeneic or allogeneic stem cell engraftment. After its systemic administration,we found saturating quantities of the antibody on all cells expressing the CD45 antigen,both in marrow and in lymphoid organs. All leukocyte subsets in peripheral blood were markedly diminished during or soon after anti-CD45 treatment,but only the effect on the lymphoid compartment was sustained. In contrast to the prolonged depletion of T and B lymphocytes from the thymus and spleen,peripheral blood neutrophils began to recover within 24 hours after the first anti-CD45 injection and marrow progenitor cells were spared from destruction,despite being coated with saturating quantities of anti-CD45. Given the transient effects of the monoclonal antibody on myelopoiesis and the more persistent effects on lymphopoiesis,we asked whether this agent could contribute to donor hematopoietic engraftment following nonmyeloablative transplantation. Treatment with anti-CD45 alone did not enhance syngeneic engraftment,consistent with its inability to destroy progenitor cells and permit competitive repopulation with syngeneic donor stem cells. By contrast,the combination of anti-CD45 and an otherwise inactive dose of total-body irradiation allowed engraftment of H2 fully allogeneic donor stem cells. We attribute this result to the recipient immunosuppression produced by depletion of CD45(+) lymphocytes. Monoclonal antibodies of this type may therefore have an adjunctive role in nonmyeloablative conditioning regimens for allogeneic stem cell transplantation. View Publication

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that,in most transplantation patients,variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype,as well as through phenotypic and functional analyses of NK cells,both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells,including patient leukemia blasts. Differently,KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly,this was due to recognition of C2 by KIR2DL2/3,as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably,however,C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether,these results may have important clinical implications for the selection of optimal donors for haplo-HSCT. View Publication

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease,leading to a speedy recovery of hematopoiesis. To meet this clinical demand,a fast MSC expansion method is required. In the present study,we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors,including stem cell factor and interleukin-3 and -6. After 8 days of culture,total cell numbers,Stro-1(+)CD44(+)CD34(-) MSCs,and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-,29-,and 8-fold,respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin,whereas markers associated with lineage differentiation,including osteocalcin (osteogenesis),type II collagen (chondrogenesis),and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis),were not detected. Upon induction,the bioreactor-expanded MSCs were able to differentiate into osteoblasts,chondrocytes,and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs. View Publication

AIM: The recently published REPAIR-AMI and ASTAMI trial showed differences in contractile recovery of left ventricular function after infusion of bone marrow-derived cells in acute myocardial infarction. Since the trials used different protocols for cell isolation and storage (REPAIR-AMI: Ficoll,storage in X-vivo 10 medium plus serum; ASTAMI: Lymphoprep,storage in NaCl plus plasma),we compared the functional activity of BMC isolated by the two different protocols. METHODS AND RESULTS: The recovery of total cell number,colony-forming units (CFU),and the number of mesenchymal stem cells were significantly reduced to 77 +/- 4%,83 +/- 16%,and 65 +/- 15%,respectively,when using the ASTAMI protocol compared with the REPAIR protocol. The capacity of the isolated BMC to migrate in response to stromal cell-derived factor 1 (SDF-1) was profoundly reduced when using the ASTAMI cell isolation procedure (42 +/- 8% and 78 +/- 3% reduction in healthy and CAD-patient cells,respectively). Finally,infusion of BMC into a hindlimb ischaemia model demonstrated a significantly blunted blood-flow-recovery by BMC isolated with the ASTAMI protocol (54 +/- 6% of the effect obtained by REPAIR cells). Comparison of the individual steps identified the use of NaCl and plasma for cell storage as major factors for functional impairment of the BMC. CONCLUSION: Cell isolation protocols have a major impact on the functional activity of bone marrow-derived progenitor cells. The assessment of cell number and viability may not entirely reflect the functional capacity of cells in vivo. Additional functional testing appears to be mandatory to assure proper cell function before embarking on clinical cell therapy trials. View Publication