Nuray Kusadasi

Successful short-term ex vivo expansion of NOD/SCID repopulating ability and CAFC week 6 from umbilical cord blood

In view of the limited potential for rapid hematological recovery after transplantation of umbilical cord blood cells (UCB) in adults, we have attempted to expand CD34+ selected hemopoietic stem cells (HSC) and progenitors in 2-week cultures of whole graft pools in the presence or absence of serum and stromal layers, and with various cytokine combinations including (1) FL + TPO; (2) FL + TPO plus SCF and/or IL6; or (3) SCF + IL6. Both in the input material and cultured grafts we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ CD38−subset by phenotyping. The highest fold-increase obtained for the number of nucleated cells (nc), CD34+, CD34+ CD38− cell numbers and CFC content was, respectively, 102 ± 76, 24 ± 19, 190 ± 202 and 53 ± 37 for stroma-free and 315 ± 110, 25 ± 3, 346 ± 410 and 53 ± 43 for stroma-supported cultures. CAFC week type 6 was maximally 11-fold expanded both under stroma-free and stroma-supported conditions. The FBMD-1 stromal cells supported a modest expansion of CD34+ CD38− cells (27 ± 18-fold) and nc (6 ± 4-fold), while a loss of CFC and CAFC subsets was observed. The stromal cells synergized with FL + TPO to give the highest expansion of hemopoietic progenitors. Stromal support could be fully replaced by complementing the FL + TPO stimulated cultures with SCF + IL6. FL + TPO were required and sufficient to give a 10- to 20-fold expansion of the ability of CD34+ UCB cells in 2-week cultures to engraft the BM of NOD/SCID mice. Stromal support, or complementation of the medium with SCF + IL6, did not significantly improve the in vivo engraftment potential. If the SRA and CAFC week 6 assays are accepted as tentative estimates of in vivo engrafting stem cells in humans, our findings may assist in the preparation of UCB grafts to meet the requirements for improved repopulation in the clinical setting.

Stromal cells from murine embryonic aorta-gonad-mesonephros region, liver and gut mesentery expand human umbilical cord blood-derived CAFC(week6) in extended long-term cultures.

The first definitive long-term repopulating hematopoietic stem cells (HSCs) emerge from and undergo rapid expansion in the embryonic aorta–gonad–mesonephros (AGM) region. To investigate the presumptive unique characteristics of the embryonic hematopoietic microenvironment and its surrounding tissues, we have generated stromal clones from subdissected day 10 and day 11 AGMs, embryonic livers (ELs) and gut mesentery. We here examine the ability of 19 of these clones to sustain extended long-term cultures (LTCs) of human CD34+ umbilical cord blood (UCB) cells in vitro. The presence of in vitro repopulating cells was assessed by sustained production of progenitor cells (extended LTC-CFC) and cobblestone area-forming cells (CAFC). The embryonic stromal clones differed greatly in their support for human HSCs. Out of eight clones tested in the absence of exogenous cytokines, only one (EL-derived) clone was able to provide maintenance of HSCs. Addition of either Tpo or Flt3-L + Tpo improved the long-term support of about 50% of the tested clones. Cultures on four out of 19 clones, ie the EL-derived clone mentioned, two urogenital-ridge (UG)-derived clones and one gastrointestinal (GI)-derived clone, allowed a continuous expansion of primitive CAFC and CFU-GM with over several hundred-fold more CAFCweek6 produced in the 12th week of culture. This expansion was considerably higher than that found with the FBMD-1 cell line, which is appreciated by many investigators for its support of human HSCs, under comparable conditions. This stromal cell panel derived from the embryonic regions may be a powerful tool in dissecting the factors mediating stromal support for maintenance and expansion of HSCs.

Stromal support augments extended long-term ex vivo expansion of hemopoietic progenitor cells.

Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+CD38− cell numbers was, respectively, 1197 and 30 937 for stroma-free and 4066 and 117 235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+CD38−CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+CD38−, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.

Stroma‐supported progenitor production as a prognostic tool for graft failure following autologous stem cell transplantation

To analyse the involvement of a possible numerical or qualitative stem cell defect in the development of sustained graft failure after autologous transplantation, we have determined the graft content of CD34+ nucleated cells, colony‐forming cells and cobblestone area‐forming cell subsets, as well as transplant ability to produce progenitors using the long‐term culture colony‐forming cell (LTC‐CFC) assay. We evaluated material from the graft reference ampoules of 13 graft failure patients after bone marrow transplantation (BMT), four graft failure patients and four isolated thrombocytopenia patients after peripheral blood stem cell transplantation (PBSCT). We compared these data with those from six successfully engrafted BMT patients and 20 engrafted PBSCT patients respectively. In the BMT setting, the LTC‐CFC 6‐week assay represented a highly significant graft failure predictor. In the PBSCT setting, the total number of 2‐week and 6‐week LTC‐CFCs transplanted per kg bodyweight (BW) showed the highest significant difference between the engrafted and the graft failure patients, as well as between the engrafted patients and the patients suffering from isolated thrombocytopenia after transplantation. These data show that the ability of a graft to generate progenitors in vitro rather than the number of primitive progenitors transplanted can have prognostic value for post‐transplant haematological reconstitution.

Predictors of short-term and long-term mortality in critically ill patients admitted to the intensive care unit following allogeneic stem cell transplantation

Historically, the mortality of patients admitted to the ICU after allogeneic stem cell transplantation (alloSCT) is high. Advancements in transplantation procedures, infectious monitoring and supportive care may have improved the outcome. This study aimed to determine short-term and long-term mortality after ICU admission of patients after alloSCT and to identify prognostic clinical and transplantation-related determinants present at ICU admission for long-term outcome. A multicenter cohort study was performed to determine 30-day and 1-year mortality within 2 years following alloSCT. A total of 251 patients were included. The 30-day and 1-year mortality was 55% and 80%, respectively. Platelet count <25 × 109/L (OR: 2.26, CI: 1.02–5.01) and serum bilirubin >19 μmol/L (OR: 2.47 CI: 1.08–5.65) at admission, other donor than a HLA-matched-related or HLA-matched-unrelated donor (OR: 4.59, CI: 1.49–14.1) and vasoactive medication within 24 h (OR: 2.35, CI: 1.28–4.31) were associated with increased 30-day mortality. Other donor than a HLA-matched-related or HLA-matched-unrelated donor (OR: 1.9, CI: 1.13–3.19), serum bilirubin >77 (OR: 2.05, CI: 1.28–3.30) and vasoactive medication within 24 h (OR: 1.65, CI: 1.12–2.43) were associated with increased 1-year mortality. Neutropenia was associated with decreased 30-day and 1-year mortality (OR: 0.29, CI: 0.14–0.59 and OR: 0.70, CI: 0.48–0.98). Myeloablative conditioning and T cell-depleted transplantation were not associated with increased mortality.