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Circulating hematopoietic stem cells serve as novel targets for in utero gene therapy¹

BARBARA MURDOCH, LISA GALLACHER, CHRISTIAN AWARAJI, DAVID A. HESS, MICHAEL KEENEY, KAREN JAY, KRISTIN CHADWICK, S. RONAN FOWLEY, KANG HOWSON-JAN, IAN CHIN YEE, DONGMEI WU, EDWARD D. SROUR, FRASER FELLOWS^* and MICKIE BHATIA^*2

The John P. Robarts Research Institute, Developmental Stem Cell Biology, The University of Western Ontario, London, Ontario, N6A 5K8, Canada;
^* Fetal Medicine Division, St. Joseph’s Hospital and London Health Sciences Centre, London, Ontario. Canada;
Department of Medicine and Hematology, London Health Sciences Centre;
Department of Hematology, Hamilton Civic Hospital, Hamilton, Ontario, Canada; and
Department of Medicine and Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA

²Correspondence: The John P. Robarts Research Institute, Developmental Stem Cell Biology, 100 Perth Dr., London, Ontario, N6A 5K8, Canada. E-mail: mbhatia{at}rri.on.ca

SPECIFIC AIMS

In this study we examined the transduction efficiency and cell cycle status of circulating human fetal blood stem cells. We reveal that retroviral transduction of unique repopulating stem cells in human fetal circulation is superior to full gestation cord blood or adult sources and is enriched for actively cycling stem cells. Our findings indicate that active, transducible hematopoietic reconstituting cells are present in the circulation of the human fetus and represent novel target cells for future in utero gene therapy trials using autologous transplantation.

PRINCIPAL FINDINGS

1. Circulating human fetal blood repopulating stem cells can be transduced at a higher efficiency than full-term cord blood
In utero gene therapy has been proposed as a method for permanent correction of somatic disorders affecting the hematopoietic system before disease initiation. However, clinical trials using transplantation of allogenic fetal liver, bone marrow, or adult stem cells have been unsuccessful largely due to the failure of sustained hematopoietic reconstitution in fetal recipients. Human-mouse xenotransplantation provides an assay system to detect human reconstituting cells by intravenous transplantation of purified cells into immune deficient NOD/SCID mice. These primitive cells capable of repopulating the bone marrow (BM) of NOD/SCID mice have been shown to be distinct from more mature progenitors detected in vitro and have been operationally defined as a SCID repopulating cell (SRC). Human SRC were previously found to be highly enriched within a rare population of CD34+CD38-Lin- cells from both FB and CB sources. After identical procedures for ex vivo culture and retroviral exposure of purified CD34+CD38-Lin- cells, transduction of FB-SRC was greatly enhanced when compared with full-term CB-SRC experiments done in parallel (Fig. 1A ). A representative analysis of the BM of NOD/SCID mice transplanted with transduced human stem cells from FB and CB illustrates that these retrovirally modified stem cells are capable of giving rise to multiple lineages with transgene expression (Fig. 1A ). Quantitative analysis by flow cytometry revealed that threefold more human reconstituting cells were expressing the transgene (GFP+CD45+) from FB-SRC compared with CB stem cells.

View larger version (48K): [in this window] [in a new window]   Figure 1. Analysis of reporter transgene expression (GFP) in NOD/SCID mice engrafted with retrovirally transduced CB-SRC and FB-SRC. Highly purified populations of CD34+CD38-Lin- cells derived from CB and FB were cultured in the presence of supernatant from retroviral producing cells and transplanted into NOD/SCID mice. A) A representative analysis of the BM of NOD/SCID mice engrafted with CB and FB-SRC. Human cells positive for the pan-leukocyte marker CD45 were gated R1. Human R1-gated cells positive for the retroviral reporter gene GFP were gated R2 = green, whereas GFP negative cells were gated R3 = blue. Both R2 and R3 color gates were used for subsequent analysis of human R1-gated cells. B) Molecular analysis indicating the presence of the retroviral transgene in the BM of CB and FB-engrafted NOD/SCID mice. Engrafted human cells (CD45+) of multiple lineages from the BM of transplanted mice were isolated by flow cytometric sorting. The presence of quality template was assessed by human specific cart-1 PCR (top panel). Genomic DNA from lymphoid (CD19+), myeloid (CD33+), and erythroid (BFU-E from CFU assay) cells were subjected to GFP PCR and visualized on an ethidium bromide stained, electrophoresed agarose gel (middle panel). Specificity of the GFP PCR was determined by probing a Southern blot of the PCR with an independently isolated GFP fragment (bottom panel). Genomic mouse (negative for cart-1), mock-infected human SRC (positive for cart-1, negative for GFP), and a murine derived, GFP-containing, retroviral-producing cell line (negative for cart-1, positive for GFP) were used as independent controls.

Human cells (gated R1) were analyzed for transgene GFP expression. Transduced GFP positive human cells were gated R2 (green) and GFP negative human cells, representing untransduced cells, were gated R3 (blue) (Fig. 1A ). Detection of human GFP+ cells within lymphoid and myeloid differentiated lineages and more primitive CD34+ cells in vivo illustrated enhanced retroviral transduction of FB stem cells with the capacity to repopulate all blood lineages vs. stem cells derived from full gestational CB (Fig. 1A ). The proportion of GFP+ human cells of all lineages engrafting NOD/SCID mice from FB stem cells was consistently higher than CB-derived repopulating cells. Human cells (CD45+) were isolated from engrafted NOD/SCID mice to verify retroviral transduction of CB and FB repopulating cells. Genomic DNA was extracted from selected lymphoid (CD45+CD19+) and myeloid cells (CD45+CD33+) along with CD45+ selected human erythroid progenitors (BFU-E) and subjected to PCR amplification of the reporter gene. A human specific gene, CART-1, was amplified from selected cells to verify the presence and quality of the human template. CART-1 was detected from all isolated populations of human cells and was not detectable in murine sources (Fig. 1B , top panel). Provirus was detected in lymphoid, myeloid, and erythroid lineages of human engrafting progeny arising from CB and FB repopulating stem cells (Fig. 1B , middle and bottom panels), confirming that human cells capable of repopulating all lineages were transduced. Our data illustrate that the human fetal circulation contains repopulating stem cells that can be retrovirally transduced to produce transgenic progeny in vivo at efficiencies superior to postnatally derived human CB stem cells.

2. Quantitative analysis of human hematopoietic repopulating cells reveals that circulating fetal blood contains a greater number of actively cycling stem cells than full-term or adult sources
The dormant nature of human blood stem cells has been thought to be the major obstacle to successful stem cell gene therapy. CD34+Lin- cells were isolated from adult mobilized peripheral blood (M-PB) (n=4), CB (n=5), and FB (n=10) and were further separated according to their cell cycle status. Purified CD34+Lin- cells residing in active G₁ or dormant G₀ stages of the cell cycle were transplanted into NOD/SCID mice from the various human ontogenic sources. A summary of the results from these experiments is shown in Fig. 2A analyzing the BM of 86 recipient mice used to compare the frequency of human SRC detected from each source by limiting dilution analysis. With the exception of one animal engrafted with the highest dose transplanted (280,000 CD34+G₁ cells), active adult M-PB CD34+Lin- cells contained no detectable repopulating cells from 5 independent donors (Fig. 2A , bottom, left). However, consistent with the precept that human blood stem cells are inactive, dormant adult M-PB CD34+Lin- cells contained repopulating cells in four of five patients (Fig. 2A , bottom, right), where only a single NOD/SCID recipient that was transplanted with as few as 20,000 G₀ CD34+Lin- cells failed to engraft. In contrast to active adult M-PB cells, full-term CB CD34+Lin- cells contained human repopulating cells in the G₁ stage of the cell cycle (Fig. 2A , middle, left). Further analysis of G₁ CB-SRC revealed their detection in as few as 5000 to 20,000 CD34+Lin- cells.

View larger version (23K): [in this window] [in a new window]   Figure 2. Proposed model of human stem cell ontogeny and cell cycle regulation. The balance between the number of repopulating stem cells residing within G0 and G1 provides a measure of the frequency of stem cells transgressing the cell cycle and undergoing mitotic divisions while retaining reconstitution capability (stem cell self-renewal). The frequency of these cell divisions is rare and intrinsically controlled, but is more abundant earlier in human ontogeny when the initial stem cell pool needs to be established. As the ontogenic age increases, the balance of stem cells residing in G0 vs. G1 begins to favor dormant stem cell states and attenuated self-renewal of stem cells. The frequency of self-renewal is directly proportional to the retroviral transduction efficiency, establishing that human fetal development provides a unique window of opportunity for stem cell gene therapy.

Consistent with the function of quiescent adult M-PB cells, dormant CB-SRC were also detectable within the G₀ state of the cell cycle (Fig. 2A , middle, right). In full-term CB, a greater number of repopulating cells were dormant compared with those in active states (Fig. 2A ). Cell cycle status of human FB-SRC was distinct from adult, but similar to CB sources in that both G₁ and G₀ CD34+Lin- FB cells contained primitive cells with repopulating ability (Fig. 2A , top). However, in contrast to full-term CB repopulating cells, the proportion of active to dormant FB stem cells was equivalent (Fig. 2A ). A representative multilineage analysis of a NOD/SCID mouse transplanted with G₁ residing CD34+Lin- FB cells is shown in Fig. 2B , 2i . Similar to previous results, the in vivo proliferative and differentiative capacity of both active G₁ and dormant G₀ FB repopulating stem cells was found to be distinct from stem cells derived from other stages of human development. FB-SRC derived from different cell cycle stages gave rise to greater myeloid differentiation (CD33 and CD15 positive cells) than lymphoid (CD19 and CD20 positive cells) compared with CB-SRC shown in Fig. 2B , 2ii . Based on our findings, we conclude that in contrast to postnatal hematopoietic sources, which contain a majority of dormant stem cells, the proportions of dormant to active stem cells in the fetal circulation are equivalent. This suggests that cell cycle transitions (cycling activity) occur more frequently among FB stem cells compared with later stages of human hematopoietic development, thereby increasing the probability of successful retroviral transduction.

CONCLUSION

Differences in vector design, cell source, and transduction protocols make it imperative to use identical conditions in order to properly assess and compare transduction efficiencies between target stem cell sources. Our study demonstrates that not all human stem cells are restricted to a quiescent state and that human stem cells undergoing cell cycle transitions can be isolated during early human development. Reconstituting cells found in the circulation of the human fetus are more permissive to retroviral transduction and functional expression of the transgene in vivo. The nature of cycling FB stem cells is unique to this stage of human development, suggesting that cell cycle transitions of human stem cells are dictated by ontogenic mechanisms specific to stem cell source. Based on our findings, we propose a model relating cell cycle activity of hematopoietic stem cells to human development, illustrating that cell cycle transitions of stem cells is dictated by intrinsic mechanisms that are specific to stem cell ontogeny (Fig. 2 ).

Previous studies have established that diagnostic sampling of an average of 3–4 ml of fetal blood does not cause hemodynamic compromise. Using these average sample volumes and quantitative analysis to determine the frequency of FB-SRC, as many as 34 FB-SRC can be harvested, which represents a greater than 24-fold excess of FB stem cells required to theoretically allow hematopoietic reconstitution in a fetal recipient. Therefore, unlike fetal liver or fetal bone marrow derived stem cells, circulating hematopoietic reconstituting cells provide a novel, previously unexplored source of human repopulating cells for autologous retransplantation in utero after retroviral transduction. Retroviral vectors have proved to be the safest and most widely tested in the clinic, but require the target cell for transduction to be undergoing cell division. Our findings indicate that ontogenic differences that dictate cell cycle regulation in circulating fetal stem cells provide a therapeutic window during human development for stem cell gene therapy using retroviral gene transfer. We suggest that cycling stem cells residing in the fetal circulation provide novel targets for future in utero gene therapy trials that will enable autologous transplantation to overcome allogenic barriers.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0654fje ; to cite this article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0654fje

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