Functional Characterization of TPO‐Expanded CD34 ⁺ Cord Blood Cells Identifies CD34 ⁻ CD61 ⁻ Cells as Platelet‐Producing Cells Early After Transplantation in NOD/SCID Mice and rCD34 ⁺ Cells as CAFC Colony‐Forming Cells

Abstract

Transplantation of thrombopoietin (TPO)‐expanded cord blood CD34+ cells accelerates human platelet recovery in NOD/SCID mice. It is unknown which subpopulations of the TPO‐expanded cells mediate accelerated platelet recovery and bone marrow (BM) engraftment. In this study, the contribution of these subpopulations to human platelet appearance in the blood and BM engraftment was studied in NOD/SCID mice. Following transplantation of CD34−/CD61−/lineage− cells (Lin−), human platelets were detected in the blood of recipient mice from day 4. Both time to platelet recovery and blood platelet counts at 6 weeks after transplantation showed Lin− dose dependence. The Lin− population was virtually negative for lineage marker expression and lacked CD42b expression but was heterogeneous with regard to CD36 and CD38 expression, reflecting a population in transit but not fully committed toward the megakaryocyte (MK) lineage. Although no definitive phenotype could be established of the cells generating prompt platelet production and cells generating platelets 6 weeks after transplantation, this relatively heterogeneous Lin− population is prerequisite to accelerate platelet recovery in vivo. The interval to platelet recovery after transplantation of the CD34+ cells remaining after expansion (rCD34+) was similar to mice transplanted with nonexpanded CD34+ cells, although the total platelet counts and the engraftment levels in the BM were lower. Cobblestone area‐forming cell colony‐forming cells resided mostly in the rCD34+ population. The pro‐MK CD61+ cells did not contribute to human platelet recovery or engraftment in the BM. Our study shows that not all expanded cells appear critical for transplantation. These data support that functional characterization of the expanded cell populations is warranted to make future expansion protocols suitable for clinical application. STEM CELLS 2012;30:988–996

Thrombopoietin Treatment of One Graft in a Double Cord Blood Transplant Provides Early Platelet Recovery While Contributing to Long-Term Engraftment in NSG Mice

“…The studies presented in this article build on our previous short-term (6 week) engraftment studies with single CB transplants in immune-deficient mice, where we have shown that TPO treatment can improve early platelet recovery and that this recovery is mostly dependent on the TPO mediated generation of CD34 -CD61 -cells [44,51]. Importantly, in our current studies, by analyzing engraftment using donorspecific HLA antibodies in the dCB transplant setting, we could further demonstrate that this improved in vivo early platelet recovery (around day 7) was indeed originating from TPO treated CD34 + cell graft, whereas the untreated CD34 + cells formed platelets at later time points (significantly from around week 2) and dominated over the TPO cultured cells.…”

“…Blood collection and human platelet measurements were performed as described previously [47,48,51]. Briefly, human platelets were stained with noncross reactive mouse anti-human CD41-PE and human CD45 cells were stained with mouse anti-human CD45-PC7 (both Beckman Coulter).…”

“…To investigate whether BM cells harvested 20 weeks after transplantation contained human hematopoietic progenitor cells (HPC), three different in vitro assays were used; HALO assays (Hemogenix, Colorado Springs, CO), myeloid CFU cultures, and cobblestone area-forming cell (CAFC) assays as described previously [51,52] and in Supplementary Data.…”

“…+ cells with TPO, the expression of CD61 (ITGB3, GPIIIa, or beta3 integrin), which forms part of the GPIIb/IIIa (CD41/CD61) complex that binds to vWF, fibronectin, fibrinogen, and vitronectin [40,43,51] + cells in the starting population. This double negative population has been shown to be responsible for the earlier platelet recovery in in vivo models [48].…”

Expression of a Surface Antigen (MA6) by Peripheral Blood CD34+Cells Is Correlated with Improved Platelet Engraftment and May Explain Delayed Platelet Engraftment Following Cord Blood Transplantation

“…Although they may provide a more definitive assessment of MK progenitor numbers, they are time-consuming, do not allow realtime evaluation and, since the colonies are the product of the proliferation of MK progenitors, they do not allow the analysis of the MK progenitor itself. However, MK progenitors can also be characterized by their expression of a range of cell surface markers, including CD41a and CD61 [13][14][15]. Such assessments can be performed rapidly and provide the potential for real-time assessment of MK progenitor cell numbers, prospective isolation, additional analyses, and therapeutic application.…”