Browsing by Author "Cheatham, Lynn"

Now showing 1 - 3 of 3
  • Thumbnail Image
    ItemOpen Access
    Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay.
    (Blood, 2016-05-12) Shoulars, Kevin; Noldner, Pamela; Troy, Jesse D; Cheatham, Lynn; Parrish, Amanda; Page, Kristin; Gentry, Tracy; Balber, Andrew E; Kurtzberg, Joanne
    Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDH(br)]), along with viable CD45(+) or CD34(+) cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDH(br), CD34(+), and CFU content of 3908 segments over a 5-year period. ALDH(br) (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34(+) (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDH(br) content of the CBU. These results suggest that the ALDH(br) segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation.
  • Thumbnail Image
    ItemOpen Access
    Histopathologic assessment of cultured human thymus.
    (PloS one, 2020-01) Hale, Laura P; Neff, Jadee; Cheatham, Lynn; Cardona, Diana; Markert, M Louise; Kurtzberg, Joanne
    The maintenance and propagation of complex mixtures of cells in vitro in the form of native organs or engineered organoids has contributed to understanding mechanisms of cell and organ development and function which can be translated into therapeutic benefits. For example, allogeneic cultured postnatal human thymus tissue has been shown to support production of naïve recipient T cells when transplanted into patients with complete DiGeorge anomaly and other genetic defects that result in congenital lack of a thymus. Patients receiving such transplants typically exhibit reversal of their immunodeficiency and normalization of their peripheral blood T cell receptor V-beta repertoire, with long-term survival. This study was designed to assess the histopathologic changes that occur in postnatal human thymus slices when cultured according to protocols used for transplanted tissues. Results showed that as thymic organ cultures progressed from days 0 through 21, slices developed increasing amounts of necrosis, increasing condensation of thymic epithelium, and decreasing numbers of residual T cells. The architecture of the thymic epithelial network remained generally well-preserved throughout the 21 days of culture, with focal expression of cytokeratin 14, a putative biomarker of thymic epithelial cells with long-term organ-repopulating potential. All organ slices derived from the same donor thymus closely resembled one another, with minor differences in size, shape, and relative content of cortex versus medulla. Similarly, slices derived from different donors showed similar histopathologic characteristics when examined at the same culture time point. Taken together, these results demonstrate that diagnostic criteria based on structural features of the tissue identifiable via hematoxylin and eosin staining and cytokeratin immunohistochemistry can be used to evaluate the quality of slices transplanted into patients with congenital athymia.
  • Thumbnail Image
    ItemOpen Access
    T cell-depleted cultured pediatric thymus tissue as a model for some aspects of human age-related thymus involution.
    (GeroScience, 2021-06) Hale, Laura P; Cheatham, Lynn; Macintyre, Andrew N; LaFleur, Bonnie; Sanders, Brittany; Troy, Jesse; Kurtzberg, Joanne; Sempowski, Gregory D
    Human age-related thymus involution is characterized by loss of developing thymocytes and the thymic epithelial network that supports them, with replacement by adipose tissue. The mechanisms that drive these changes are difficult to study in vivo due to constant trafficking to and from the thymus. We hypothesized that the loss of thymocytes that occurs during human thymic organ cultures could model some aspects of thymus involution and begin to identify mechanisms that drive age-related changes in the thymic microenvironment. Potential mechanistically important candidate molecules were initially identified by screening conditioned media from human thymus organ cultures using antibody microarrays. These candidates were further validated using cultured tissue extracts and conditioned media. Results were compared with gene expression studies from a panel of well-characterized (non-cultured) human thymus tissues from human donors aged 5 days to 78 years. L-selectin released into conditioned media was identified as a biomarker for the content of viable thymocytes within the cultured thymus. Levels of the chemokines CCL21 and CXCL12, likely produced by surviving thymic epithelial cells, increased markedly in conditioned media as thymocytes were lost during culture. Native non-cultured thymus from adults older than 18 years also showed a strong trend toward increased CCL21 expression, in conjunction with significant decreases in thymocyte-related mRNAs compared with thymus from subjects younger than 18 years. Together, these findings demonstrate that use of postnatal human thymus organ cultures can model some aspects of human age-related thymic involution.