Lisa Gallacher

Human Embryonic Stem Cells Possess Immune-privileged Properties

Human embryonic stem cells (hESCs) are envisioned to be a major source for cell‐based therapies. Efforts to overcome rejection of hESCs include nuclear transfer and collection of hESC banks representing the broadest diversity of major histocompatability complex (MHC) polymorphorisms. Surprisingly, immune responses to hESCs have yet to be experimentally evaluated. Here, injection of hESCs into immune‐competent mice was unable to induce an immune response. Undifferentiated and differentiated hESCs failed to stimulate proliferation of alloreactive primary human T cells and inhibited third‐party allogeneic dendritic cell‐mediated T‐cell proliferation via cellular mechanisms independent of secreted factors. Upon secondary rechallenge, T cells cocultured with hESCs were still responsive to allogeneic stimulators but failed to proliferate upon re‐exposure to hESCs. Our study demonstrates that hESCs possess unique immune‐privileged characteristics and provides an unprecedented opportunity to further investigate the mechanisms of immune response to transplantation of hESCs that may avoid immune‐mediated rejection.

Wnt-5A augments repopulating capacity and primitive hematopoietic development of human blood stem cells in vivo

Human hematopoietic stem cells are defined by their ability to repopulate multiple hematopoietic lineages in the bone marrow of transplanted recipients and therefore are functionally distinct from hematopoietic progenitors detected in vitro. Although factors capable of regulating progenitors are well established, in vivo regulators of hematopoietic repopulating function are unknown. By using a member of the vertebrate Wnt family, Wnt-5A, the proliferation and differentiation of progenitors cocultured on stromal cells transduced with Wnt-5A or treated with Wnt-5A conditioned medium (CM) was unaffected. However, i.p. injection of Wnt-5A CM into mice engrafted with human repopulating cells increased multilineage reconstitution by >3-fold compared with controls. Furthermore, in vivo treatment of human repopulating cells with Wnt-5A CM produced a greater proportion of phenotypically primitive hematopoietic progeny that could be isolated and shown to possess enhanced progenitor function independent of continued Wnt-5A treatment. Our study demonstrates that Wnt-5A augments primitive hematopoietic development in vivo and represents an in vivo regulator of hematopoietic stem cell function in the human. Based on these findings, we suggest a potential role for activation of Wnt signaling in managing patients exhibiting poor hematopoietic recovery shortly after stem cell transplantation.

Human embryonic-derived hematopoietic repopulating cells require distinct factors to sustain in vivo repopulating function.

OBJECTIVE We have previously identified a novel circulating embryonic blood cell capable of pluripotent hematopoietic reconstitution, which may serve as a target for in utero stem cell therapy. Based on its unique biological properties and ontogenic origin, we aim to examine the ability to maintain and retrovirally transduce fetal blood (FB) reconstituting cells in ex vivo culture conditions previously optimized for pluripotent hematopoietic repopulating cells derived from later stages of human ontogeny. METHODS FB cells were evaluated for proliferative potential, progenitor composition, and SCID-repopulating cell (SRC) capacity before and after 3 days of serum free (SF) ex vivo culture using the previously optimized growth factor conditions of SCF, Flt-3L, IL-3, IL-6, and G-CSF (GF Mix), in comparison to cultures using GF Mix + oncostatin M (OSM), or SCF + Flt-3L. We further examined the ability to retrovirally transduce FB-SRC maintained in culture using SCF + Flt-3L alone. RESULTS Circulating FB-SRC could not be maintained under GF Mix conditions previously shown to sustain CB (cord blood)-SRC. Ex vivo culture with SCF + Flt-3L reduced the proliferation of primitive FB cells lacking lineage commitment markers (Lin(-)), but expanded FB progenitors and sustained FB-SRC compared to culture with GF Mix with and without OSM. Using SCF + Flt-3L, FB-SRC capable of multilineage reconstitution were successfully transduced, suggesting that SCF and Flt-3L are necessary and sufficient for the survival and transduction of human hematopoietic repopulating cells of embryonic origin. CONCLUSION Our study provides novel insights into the requirements of primitive FB reconstituting cells that are essential for developing in utero stem cell gene therapy protocols, and further illustrates the biological distinctiveness of FB-SRC compared to hematopoietic repopulating cells from other stages of human ontogeny.

Undetectable leukemic blasts and absence of NOD/SCID leukemia-initiating cells in cord blood from a case of maternal AML

Undetectable leukemic blasts and absence of NOD/SCID leukemia-initiating cells in cord blood from a case of maternal AML