Molly Nelson-Holte

Thymidine analogs are transferred from prelabeled donor to host cells in the central nervous system after transplantation: a word of caution

Thymidine analogs, including bromodeoxyuridine, chlorodeoxyuridine, iododeoxyuridine, and tritiated thymidine, label dividing cells by incorporating into DNA during S phase of cell division and are widely employed to identify cells transplanted into the central nervous system. However, the potential for transfer of thymidine analogs from grafted cells to dividing host cells has not been thoroughly tested. We here demonstrate that graft‐derived thymidine analogs can become incorporated into host neural precursors and glia. Large numbers of labeled neurons and glia were found 3–12 weeks after transplantation of thymidine analog‐labeled live stem cells, suggesting differentiation of grafted cells. Remarkably, however, similar results were obtained after transplantation of dead cells or labeled fibroblasts. Our findings reveal for the first time that thymidine analog labeling may not be a reliable means of identifying transplanted cells, particularly in highly proliferative environments such as the developing, neurogenic, or injured brain.

Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40–80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 103-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Multipotent adult progenitor cells sustain function of ischemic limbs in mice.

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.

Reversal of hyperglycemia by insulin-secreting rat bone marrow- and blastocyst-derived hypoblast stem cell-like cells.

β-cell replacement may efficiently cure type 1 diabetic (T1D) patients whose insulin-secreting β-cells have been selectively destroyed by autoantigen-reactive T cells. To generate insulin-secreting cells we used two cell sources: rat multipotent adult progenitor cells (rMAPC) and the highly similar rat extra-embryonic endoderm precursor (rXEN-P) cells isolated under rMAPC conditions from blastocysts (rHypoSC). rMAPC/rHypoSC were sequentially committed to definitive endoderm, pancreatic endoderm, and β-cell like cells. On day 21, 20% of rMAPC/rHypoSC progeny expressed Pdx1 and C-peptide. rMAPCr/HypoSC progeny secreted C-peptide under the stimulus of insulin agonist carbachol, and was inhibited by the L-type voltage-dependent calcium channel blocker nifedipine. When rMAPC or rHypoSC differentiated d21 progeny were grafted under the kidney capsule of streptozotocin-induced diabetic nude mice, hyperglycemia reversed after 4 weeks in 6/10 rMAPC- and 5/10 rHypoSC-transplanted mice. Hyperglycemia recurred within 24 hours of graft removal and the histological analysis of the retrieved grafts revealed presence of Pdx1-, Nkx6.1- and C-peptide-positive cells. The ability of both rMAPC and HypoSC to differentiate to functional β-cell like cells may serve to gain insight into signals that govern β-cell differentiation and aid in developing culture systems to commit other (pluripotent) stem cells to clinically useful β-cells for cell therapy of T1D.

Late CD8+ T cell-dependent xenoantibody production in innate tolerant nude rats after hamster islet grafting but not after hamster heart grafting.

Background. Xenograft rejection can be provoked by both the innate and adaptive immune compartments and close reciprocal interactions exist between these two systems. We investigated the interdependent roles of T and B lymphocytes in vascularized (heart) and cellular (islet) xenograft rejection in a model with established xeno-nonreactivity of the innate immune system. Methods. Specific innate xenotolerance was induced in nude rats bearing either a hamster heart or a hamster pancreatic islet graft by a tolerizing regimen consisting of donor antigen infusion, temporary natural killer cell depletion and a 4-week administration of leflunomide. One month after transplantation, syngeneic CD4+ and CD8+ T cells were adoptively transferred. Results. Both vascular and cellular xenografts were rejected after CD4+ T cell reconstitution, corresponding with production of high IgM and IgG xenoantibody titers. Deposition of xenoantibodies and complement was seen in the heart but not in the islet xenografts. After infusion of CD8+ T cells, xenohearts underwent a delayed type of rejection without xenoantibody production and xenoislets were not rejected. In xenoislet recipients, CD8+ dependent B cells were not tolerized, resulting in the production of IgG xenoantibodies belonging to Th2-dependent isotypes, known not to cause graft rejection, and deposited at the graft implantation site. Conclusions. We conclude that distinct mechanisms of immune activation underlie xenogeneic reactions against vascular and cellular grafts.