Quantitative Analysis Demonstrates Expansion of SCID-Repopulating Cells and Incr - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

a Stem Cell Biology Laboratory, Large Scale Biology Corporation, Vacaville, California, USA;
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b Division of Hematology/Oncology, University of California at Davis Cancer Center, Sacramento, California, USA;  j+ V- A6 Z" ^9 s# {& z" n9 X

c Jackson Laboratories, Sacramento, California, USA;( |, v% c) a! _  x1 ~

d University of California at Davis Medical Center, Department of Pathology, Davis, California, USA5 n) t. }: X8 W; l/ \& S9 M0 k
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Key Words. Cord blood ? SCID-repopulating cells ? Ex vivo expansion ? Endothelial cells
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John P. Chute, M.D., Stem Cell Transplantation Program, Duke University, 2400 Pratt Street, Suite 1100, Durham, North Carolina 27710, USA. Telephone: 919-668-1011; Fax: 919-668-1091; e-mail: johnchute@duke.edu; E9 Y( m! L/ h0 Y! D3 |

ABSTRACT

Umbilical cord blood (CB) is an attractive source of hematopoietic stem cells for transplantation due to its high concentration of stem/progenitor cells , lower risk for graft-versus-host disease , and relative availability and ease of procurement . However, clinical application of CB for transplantation has been limited by the low absolute stem cell numbers per graft , risk of graft failure , and slower platelet and neutrophil engraftment compared with other sources of adult stem cells . A rationale has, therefore, existed to attempt ex vivo expansion of CB stem cells for application in larger-sized children and adults  and to accelerate the hematopoietic and immune system recovery following transplantation .! P9 \4 f  v: i
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In the steady state, the CD34 CD38- population is enriched for primitive hematopoietic cells capable of repopulating nonobese diabetic severe combined immunodeficient (NOD/SCID) mice (SCID-repopulating cells ) . However, following ex vivo culture, dissociation between CD34 CD38- cell expansion and SCID-repopulating capacity has been observed . Therefore, investigators have focused on the SCID-repopulating capacity of cultured hematopoietic cells rather than the phenotype as a measure of stem cell frequency . Recent preclinical studies have indicated that human CB CD34  cells can be expanded in vitro under various conditions for up to 1–10 weeks . Liquid suspension culture conditions, including stem cell factor (SCF), Flt-3 ligand, and thrombopoietin (TPO), have also been reported to optimize the ex vivo maintenance of SRCs within human CB . However, a recent study suggested that ex vivo expansion of human CB stem cells in stroma-free conditions may not maintain nor expand long-term repopulating cells as effectively as previously hypothesized .: L  u( @/ n$ T2 d

That study demonstrated that CB stem cells cultivated in vitro with megakaryocyte growth and development factor (MGDF) SCF G-CSF were unable to provide long-term repopulation in a fetal sheep model . As importantly, those investigators also demonstrated, in a phase I clinical trial, that transplantation of unrelated donor CB CD34  cells, which were cultured for 10 days with SCF G-CSF MGDF, did not result in faster in vivo hematopoietic recovery as compared with historical unmanipulated CB transplant controls . Jaroscak et al. also showed that infusion of ex vivo expanded CB cells cultured under stroma-free conditions with PIXY 321 (GM-CSF-interleukin -3 fusion protein) Flt-3 ligand erythropoietin did not impart a faster recovery of either platelets or neutrophils compared with historical controls .1 X8 t$ @% C, M
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The hematopoietic capacity of endothelial cells has been suggested by the interdependent development of vascular endothelial precursors and hematopoietic progenitors during embryogenesis . Adult bone marrow (BM) endothelial cells have also been shown to support the proliferation of committed myeloid and megakaryocytic progenitors in vitro . We previously demonstrated that coculture with a porcine brain microvascular endothelial cell line supported the expansion of adult human BM CD34 CD38- cells as well as cells capable of providing long-term hematopoietic recovery in lethally irradiated baboons . More recently, we showed that short-term coculture with primary human brain endothelial cells (HUBECs) caused a 4.1-fold expansion of SRCs within adult human BM . Since ex vivo expansion of CB stem cells has therapeutic potential in the transplantation of adult patients, we subsequently tested whether contact or noncontact HUBEC cultures could result in a high frequency of SRCs within human CB compared with liquid suspension cultures. Using a quantitative limiting dilution analysis, we demonstrated that HUBEC coculture led to a greater SRC frequency as compared with fresh CB CD34  cells or liquid suspension-cultured cells. Remarkably, cell-to-cell contact with HUBECs not required for SRC expansion to occur, indicating that HUBEC-secreted soluble factors accounted for this effect. These data suggest that HUBEC-based expansion protocols have the potential to improve upon current methods to expand CB stem cells for clinical transplantation.
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MATERIALS AND METHODS- j+ L* r% w6 B! Q$ Y
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HUBEC Coculture Supported the Expansion of CB CD34  Cells and Colony-Forming Cells

Primary HUBECs demonstrated a cobblestone morphology in culture, and >90% expressed human von Willebrand factor, as previously described . The effects of liquid suspension culture, HUBEC coculture, and noncontact HUBEC culture on the in vitro expansion of CB CD34  cells and colony-forming cells (CFCs) were compared. All cultures were supplemented with TSF, as noted in Materials and Methods. HUBEC coculture supported a 26-fold increase in total cells, a 19-fold increase in CD34  cells, and a 156-fold increase in CD34 CD38- cells after 7 days (Table 1). As shown in a representative experiment (Fig. 1), CB CD34 CD38- cells increased from a mean 11.2% of the day-0 CB CD34  population to 66.9% of the total population at day 7 (Fig. 1A, 1B). Similarly, HUBEC noncontact cultures supported a 32-fold increase in total cells, a 14-fold increase in CD34  cells, and a 115-fold increase in CD34 CD38- cells (Fig. 1C). In contrast, liquid suspension cultures supplemented with TSF supported a 16-fold increase in total cells, but we observed a decline in the percentage of CD34  and CD34 CD38- cells, resulting in a 5.4-fold increase in CD34  cells and a 10-fold increase in CD34 CD38- cells by day 7 (Fig. 1D).+ |/ k: o+ e* \2 z+ T

Table 1. HUBEC coculture supports greater expansion of human CB progenitor cells

Figure 1. Phenotypic analysis of CB CD34  cells at day 0 and following ex vivo culture. Purified human CB CD34  cells were plated on confluent HUBEC monolayers, noncontact HUBEC cultures, and liquid suspension cultures in the presence of optimal concentrations of TPO SCF Flt-3 ligand. A) The phenotype of untreated CB CD34  cells at day 0. B) HUBEC-cultured CB cells at day 7, demonstrating a high percentage of CD34 CD38- cells. C) Noncontact HUBEC-cultured CB cells at day 7, showing preservation of the CD34 CD38- phenotype. D) Liquid suspension cultures, demonstrating a decline in CD34  and CD34 CD38- cells at day 7. Log fluorescence distribution of CD34 expression is shown along the x-axis, and CD38 expression along the y-axis. Isotype matched control monoclonal antibody staining is shown at the left of each figure. The numbers within these quadrants indicate the percentage of total cells that fall within the particular quadrant.; p+ h6 I6 `/ M7 J0 G- Z
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HUBEC coculture induced a 73-fold increase in colony-forming units-granulocyte-macrophage (CFU-GM), an 11-fold increase in BFU-E, and a 23-fold increase in CFU-mix compared with day-0 CB CD34  cells (Table 1). HUBEC noncontact cultures supported a comparable expansion of total CFCs, with a greater proportion of BFU-E than contact HUBEC cultures. Liquid suspension cultures also induced expansion of CFCs, but the expansion of CFU-GM and CFU-total was less than 50% of the expansion observed following HUBEC coculture." ]& g! h/ y1 Z
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HUBEC Coculture Increased the SCID-Repopulating Capacity of CB CD34  Cells

NOD/SCID mice were transplanted with either fresh CB CD34  cells (n = 37 mice), CB CD34  cells cultured with TSF (n = 45 mice), or HUBEC-cultured cells (n = 46 mice) over a range of doses designed to achieve nonengraftment in a fraction of mice. As shown in Figure 2A, transplantation of 1 x 103 fresh CB CD34  cells resulted in no engraftment in eight mice. Similarly, the progeny of 1 x 103 CB CD34  cells cultured with TSF also failed to engraft in 10 mice. The progeny of 1 x 103 CB CD34  cells cultured with HUBECs engrafted in 1 of 10 mice (10%) (Fig. 2A). Over a dose range of 5 x 103 to 1 x 104 cells, fresh CB CD34  cells engrafted in only 4 of 16 mice (25%), whereas the progeny of 5 x 103 to 1 x 104 CB CD34  cells cultured with TSF engrafted in 9 of 19 mice (47%). In contrast, over the same dose range, the progeny of HUBEC-cultured CB CD34  cells engrafted in 14 of 20 mice (70%) (Fig. 2B, 2C(John P. Chutea,b, Garrett)

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