Hematopoietic stem cells (HSCs) possess the unique capacity to self-renew and give rise to all types of mature cells within the blood and immune systems. Despite our progress in understanding the molecular factors that support the self-renewal and differentiation of the hematopoietic system in vivo, less is known on how to modulate the factors that govern the self-renewal of hematopoietic stem/progenitor cells (HSPCs) ex vivo. Unlike in the case of embryonic stem (ES) cells, expansion of CD34+ HSPC in culture in general is at the expense of loss of “stemness”.
HSPCs can be collected from cord blood (CB), mobilized peripheral blood (PBSC), and rarely bone marrow (BM) at the present practice. Due to the limited CD34+ cell number in one single cord blood unit, much of the current efforts on developing technology of ex vivo expansion of HSPC uses cord blood as a source and is clinically applied to cord blood HSPC transplants. However, there are growing needs for expanding PBSCs for transplant-related practices such as HSPC expansion from poor autologous mobilizations, gene therapy or genome-editing via TALENs or CRISPR/Cas9. Developing a technology that would allow HSPC ex vivo expansion from both CB and PBSC sources is a key step towards this goal.
Several groups have reported that ex vivo culture of CB CD34+ cells with HDAC inhibitors (HDACi) can lead to expansion of a CD34+CD90+ population, which is responsible for enhanced marrow-repopulating potential. In this study, we ask whether HDACi can have a similar effect on PBSC CD34+ cells. Furthermore, we have explored the mechanism(s) mediated by HDACi in CD34+CD90+ cell expansion.
First we assessed a panel of HDACi to identify the most potent molecule for CD34+CD90+ cell expansion and selected trichostatin A (TSA) for future study. Next, TSA was added to the cytokines (SCF, Flt3 ligand, IL-3 and IL-6) to further characterize its potential in PBSC CD34+CD90+ cell expansion. We observed TSA treated CD34+ cultures with cytokines yielded 4.8 times greater numbers of CD34+CD90+ cells as compared to the cultures containing cytokines with DMSO solvent control. We next examined SCID repopulating ability (SRA) to evaluate the cultured CD34+CD90+ cells in vivo. We observed that mice transplanted with 3 million CD34+ cells treated with TSA had higher degree of human cell chimerism than those treated with DMSO at 8 weeks bone marrow and peripheral blood (32% vs 18%; p < 0.05), which was further confirmed by secondary transplantation. Furthermore, these cells were capable of differentiating into cells belonging to multiple hematopoietic lineages.
To investigate the molecular mechanisms responsible for the expansion of functional HSCs and progenitors that were observed following TSA treatment, we analyzed the expression levels of several HSPC related genes, which were compared between CD34+ cells treated with TSA and DMSO. Significantly higher transcript levels were detected for GATA 2 (p < 0.05), HOXB4 (p < 0.05), HOXA9 (p < 0.05), and SALL4 (p < 0.05) by real time quantitative RT-PCR in TSA expanded cells as compared with controls.
To evaluate whether these transcription factors can contribute to the expansion of CD34+CD90+ cells, GATA2, HOXB4 or SALL4 shRNAs were transfected into PBSC CD34+ cells, followed by culture with TSA. Among these transcription factors, knocking down SALL4 expression led to the most significant reduction of CD34+CD90+ cell numbers (33% of reduction). In addition, overexpression of SALL4 in PBSC CD34+ cells led to an increase of CD34+CD90+ cell numbers when compared to controls (p < 0.05).
Overall, our study demonstrated a novel HDACi mediated ex vivo PBSC culture technology that leads to the expansion of CD34+CD90+ cells and an increase of the marrow repopulating potential of these cells. Both gain-of-function and loss-of-function studies support that SALL4 is a key transcription factor responsible for the process. Future study on the use of HDACi or other methods to increase SALL4 expression/function will be highly beneficial to ex vivo HSPC (CB and PBSC) expansion technology.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.