Here, we have used in vivo bioluminescent imaging to compare the engraftment potential of human cord blood CD34+ cells expanded in culture for 10 days with the equivalent unexpanded fraction. Our results validate the hypothesis that expanded UCB progenitors can produce durable, multilineage engraftment, nearly comparable with that of unexpanded UCB progenitors.
A recent publication by Giassi et al. [45
] demonstrated that cultured human UCB cells could generate myeloid and erythroid lineages, but not lymphoid lineage cells in NSG mice unless the mice were pretreated with tumor necrosis factor α. In our model, the majority of detectable human cells were CD19+
following the onset of engraftment; however, as early as 8 weeks post-transplant, we were able to detect CD3+
human cells as well. Furthermore, by 10 months post-transplant, a majority of cells in the periphery were CD3+
. Although the only physical difference between our expansion protocol and that of Giassi et al. [45
] is the use of G-CSF, this cytokine is known to promote the enhancement of myeloid, not lymphoid, lineages. Given this, it is unlikely that this modification to our expansion protocol contributed significantly to our differential results. More likely, Giassi et al. [45
] did not report results that extended beyond 8 weeks post-transplant. Because of the time that it might take for T cells to mature in the thymus and for the thymus itself to become populated with de novo-generated human dendritic cells, it is unlikely that T cells would be observed prior to this time point. Indeed, we ourselves did not observe the development of T cells until the later stages of engraftment. The shift from a predominantly CD19+
lymphoid subset to a predominantly CD3+
subset approximately 10 months post-transplant was curious. Preliminarily, we have hypothesized that this shift may have been a result of physiologic events related to aging and not necessarily a result of events that were hematopoietic in nature. Histologic analysis of the long bones revealed an abnormal cellular distribution. Although we found human cells in the epiphysis, the diaphysis was calcified and completely a cellular, suggesting that the ability of older NSG mice to produce B cells could be significantly impaired. However, we did not examine untransplanted controls, and we cannot rule out the hypothesis that the grafts themselves were the cause of the diaphysis calcification.
Our lentiviral transduction efficiency was consistent with that reported in a recent publication by Liu et al. [46
], which outlines the major parameters for efficient lentiviral transduction and engraftment of human CD34+
UCB. Liu [46
] demonstrated that optimal transduction and engraftment efficiency is achieved by transducing unstimulated stem cells for 5 hours followed by 3 days of culture. We also observed the importance of incorporating a short culture period subsequent to an overnight transduction. In order to allow the viral vector to survive an overnight incubation period, it would have been necessary to replace the RD114 viral envelope coat protein with vesicular stomatitis virus G (VSV-G). Though VSV-G is able to more efficiently transduce CD34+
stem cells, it is also much more toxic, and cell viability following a VSV-G transduction is unacceptable. Because RD114 is not able to form viable virus particles with any third-generation, self-inactivated lentiviral vectors, we did all the described studies with first-generation vectors. Such a protocol is fine for in vitro studies, but would be unacceptable in a clinical setting. If we are to use our vectors clinically, we will likely have to adopt a transduction period similar to that of Liu [46
], though they did not demonstrate the potential of their transduced cells to mediate long-term engraftment.
The sensitivity of luciferase-based bioluminescent imaging is much greater than that of engraftment screening assays that detect the presence of human cells in peripheral blood. Although we were able to detect clear signals of engraftment in the marrow spaces no later than 10 days post-transplant and predict successful engraftment as early as day 6, we were unable to detect human cells in the periphery by flow cytometry until post-transplant day 28 at the earliest. Although bioluminescent imaging is not applicable clinically, we show, in principle, that engraftment failure can be detected very early. We determined that transduction of as few as 3% of cells could still result in a detectable image (data not shown), and we speculate that the labeling of patient products prior to transplantation could conceivably have predictive merit in a number of clinical protocols if used in conjunction with appropriate technologies that allow imaging of cells within human recipients.
In our imaging study, we were able to detect hematopoiesis in a defined area within the calvarium. This area is not generally appreciated to be hematopoietically active; however, a recent report by Lo Celso et al [47
]. demonstrates the importance of the calvarium as a focus of hematopoietic activity. By imaging, we demonstrated that UCB cells migrate to a specific calvarial focus and expand significantly (per available volume niche). This area, which does not exist at the time of birth, develops during the postnatal period and may sustain unique hematopoietic and niche migration characteristics. At 2 weeks post-transplant, there were significant differences in the calvarial engraftment signal between unexpanded and expanded HSC grafts. With time, this difference diminished, and no significant differences were observed long term. The significance of this phenomenon is unclear and requires further study. It is our hypothesis that stem cells lose the ability to migrate to the calvarium during expansion, and that expanded grafts can only populate the calvarium after de novo hematopoiesis occurs at other sites, that is, in the long bones or the spine. If this is indeed the case, a better understanding of the mechanism of calvarial engraftment could conceivably improve the rate of engraftment in expanded UCB clinical protocols.
In summary, with the exception of a small delay in early engraftment, human UCB cells expanded 10 days in culture could engraft and differentiate into multiple hematopoietic lineages in NSG mice in virtually the same manner as unexpanded human UCB cells. The ability to image transduced stem cells might allow the prediction of engraftment failure a few days after transplant, allowing the clinical decision-making process to be significantly enhanced.