Differential effects of HOXB4 on nonhuman primate short- and long-term repopulating cells

Abstract

Background: Hematopoietic stem cells (HSCs) or repopulating cells are able to self-renew and differentiate into cells of all hematopoietic lineages, and they can be enriched using the CD34 cell surface marker. Because of this unique property, HSCs have been used for HSC transplantation and gene therapy applications. However, the inability to expand HSCs has been a significant limitation for clinical applications. Here we examine, in a clinically relevant nonhuman primate model, the ability of HOXB4 to expand HSCs to potentially overcome this limitation.

Methods and findings: Using a competitive repopulation assay, we directly compared in six animals engraftment of HOXB4GFP (HOXB4 green fluorescent protein) and control (yellow fluorescent protein [YFP])-transduced and expanded CD34+ cells. In three animals, cells were infused after a 3-d transduction culture, while in three other animals cells were infused after an additional 6-9 d of ex vivo expansion. We demonstrate that HOXB4 overexpression resulted in superior engraftment in all animals. The most dramatic effect of HOXB4 was observed early after transplantation, resulting in an up to 56-fold higher engraftment compared to the control cells. At 6 mo after transplantation, the proportion of marker gene-expressing cells in peripheral blood was still up to 5-fold higher for HOXB4GFP compared to YFP-transduced cells.

Conclusions: These data demonstrate that HOXB4 overexpression in CD34+ cells has a dramatic effect on expansion and engraftment of short-term repopulating cells and a significant, but less pronounced, effect on long-term repopulating cells. These data should have important implications for the expansion and transplantation of HSCs, in particular for cord blood transplantations where often only suboptimal numbers of HSCs are available.

Figure 1. Efficient HOXB4-Mediated Ex Vivo Expansion of M. nemestrina CD34 ⁺ Cells

The mean and standard deviation of four independent experiments are shown. HOXB4GFP- or YFP (control)–transduced M. nemestrina bone marrow CD34 ⁺ cells were kept in liquid culture for 4 wk. Culture media were changed regularly to keep cells at optimum densities. (A) Percentage of HOXB4GFP ⁺ or YFP ⁺ cells. (B) Fold expansion of total nucleated cells. (C) Fold expansion of CFCs.

Figure 2. Improved Engraftment of HOXB4GFP-Transduced CD34 ⁺ Cells

Equal numbers of macaque CD34 ⁺ cells were transduced in 3-d transduction cultures with either the HOXB4GFP- or YFP-expressing vector. Transduced cells were infused into myeloablated animals. The percentage of HOXB4GFP ⁺ and YFP ⁺ granulocytes was assessed by flow cytometry. Shown is the engraftment of HOXB4GFP ⁺ and YFP ⁺ granulocytes after transplantation.

Figure 3. Improved Engraftment of HOXB4GFP-Transduced and HOXB4GFP-Expanded CD34 ⁺ Cells

Equal numbers of macaque CD34 ⁺ cells were transduced in 3-d transduction cultures with either the HOXB4GFP or YFP vector and then cultured for an additional 9 d (T02266) or 6 d (K03290 and J02152) in the presence of SCF, TPO, Flt-3L, and G-CSF. All the transduced and expanded cells were infused into myeloablated animals. The percentage of HOXB4GFP ⁺ and YFP ⁺ granulocytes was assessed by flow cytometry. Shown is the engraftment of HOXB4GFP ⁺ and YFP ⁺ granulocytes after transplantation. (A) T02266, (B) K03290, and (C) J02152.

Figure 4. HOXB4-Mediated Expansion Results in Polyclonal Repopulation

LAM-PCR was performed on lysates of sorted peripheral blood cells from K03290 at 3 mo post-transplantation. The lane on the far left is a 25-bp DNA ladder. HOXB4GFP indicates a triplicate LAM-PCR analysis for DNA from HOXB4GFP ⁺ cells. YFP indicates a triplicate LAM-PCR analysis for DNA from YFP ⁺ cells.

Figure 5. Differential Effects of HOXB4 Overexpression on Granulocyte and Lymphocyte Engraftment and Marking Levels in Platelets and Red Blood Cells

(A) Flow-cytometric analysis of CD13 ⁺, CD14 ⁺, CD3 ⁺, and CD20 ⁺ cells in peripheral blood of M01035, K00339, and K03290 at 3 and 6 mo after transplantation. (B) HOXB4GFP or YFP marking levels of FACS-sorted CD13 ⁺ myeloid cells, CD14 ⁺ monocytes, CD3 ⁺ T cells, and CD20 ⁺ B cells from peripheral blood of M01035 and K00339 at 3 or 6 mo after transplantation as determined by Taqman PCR. (C) Representative flow-cytometric analysis of marking levels in platelets and red blood cells at 3 mo after transplantation for M01035, K00339, and K03290.

Figure 6. HOXB4 Protein Expression in HOXB4-Marked Cells

(A) HOXB4-transduced CD34 ⁺ cells (lane 1) or FACS-sorted HOXB4GFP ⁺ bone marrow cells from M01035 at 4 mo (lane 2) or 10 mo post-transplantation (lane 3) were subjected to Western blot for detection of HOXB4 expression levels. (B) HOXB4GFP ⁺ or YFP ⁺ cells from bone marrow or peripheral blood in M01035 were isolated and subjected to Western blot analysis. Protein lysate of peripheral blood cells from a normal control animal was used as a negative control.