Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:00 |只看该作者 |倒序浏览 |打印

作者：Takahiro Suzukia,b, Yasuhisa Yokoyamac, Keiki Kumanod, Minoko Takanashie, Shiro Kozumaf, Tsuyoshi Takatob,g, Tatsutoshi Nakahatah, Mitsuo Nishikawai, Seiji Sakanoj, Mineo Kurokawac, Seishi Ogawaa,b, Shigeru Chibad作者单位：aDepartment of Regeneration Medicine for Hematopoiesis, Graduate School of Medicine, University of Tokyo, Tokyo, Japan;bDivision of Tissue Engineering, University of Tokyo Hospital, Tokyo, Japan;cDepartment of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan;dD * Z3 |) ]) T; [: @  d* M9 a' |  I

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      【摘要】( U! H0 X: z# U1 b
   Correspondence: Shigeru Chiba, M.D., Ph.D., Department of Cell Therapy and Transplantation Medicine, University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Telephone: 81-3-5804-6263; Fax: 81-3-5804-6261; e-mail: schiba-tky@umin.ac.jp or Seishi Ogawa, M.D., Ph.D., Department of Regeneration Medicine for Hematopoiesis, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Telephone: 81-3-3815-5411, ext. 35609; Fax: 81-3-5804-6261; e-mail: sogawa-tky@umin.ac.jp
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Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long-term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133-sorted cells using a soluble form of Delta1. After a 3-week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt-3 ligand, interleukin (IL)-3, and IL-6/soluble IL-6 receptor chimeric protein (FP6) in a serum- and stromal cell-free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self-renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/cnull mice, which showed myeloid, B, T, and natural killer cells as well as CD133 CD34  cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133-sorted cells contained approximately 4.5 times more SRCs than the CD34-sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.
      【关键词】 AC antigen Hematopoietic stem cells Notch Stem cell expansion% T) l. x) v8 w. r
   INTRODUCTION# w: C/ z# d( f  G

Umbilical cord blood (CB) is an established stem cell source for hematopoietic stem cell (HSC) transplantation. In many cases, however, CB transplantation is unavailable to patients with relatively high body weight because of the insufficient number of HSCs obtained from a single CB unit .% x. R# i8 W+ ^/ X# ]' s

Another possibility to acquire a higher number of stem cells is ex vivo expansion of HSCs. Although many reports have described potential methods to increase HSCs ex vivo, only a few of them have clearly demonstrated the expansion of long-term severe combined immunodeficient (SCID) repopulating cells (SRCs), currently the only reliable measure of HSCs , might be a promising agent for ex vivo expansion of HSCs.% [9 {. R4 Y* a: @& |: o$ H, C2 H

Another method that is potentially useful for stem cell expansion is the use of Notch signaling. It is mediated by interactions between transmembrane receptors (Notch1, -2, -3, and -4) and their membrane-bound ligands (Delta and Jagged family molecules). The signaling pathway is known to have differentiation-inhibitory effects in different stem cell systems, including hematopoiesis . These findings strongly prompt us to use Notch ligands in combination with FP6, for stem cell expansion.
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The initial stem cell source is also an important issue for obtaining the maximum efficiency of stem cell expansion. Whereas many investigators use the CD34-sorted cells as a source of stem cell expansion, recent reports suggested that CD133 sorting can concentrate SRCs more efficiently than CD34 sorting , and it is still open to question which population is more suitable for stem cell expansion.% Y0 r, @' Q" \
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In this study, we first addressed the issue of stem cell sources, demonstrating that the CB CD133-sorted cells contained an approximately 4.5-fold greater absolute number of SRCs than CD34-sorted cells. We next evaluated the integrated effect of Notch and gp130 signalings using soluble Delta1 and FP6 in combination with SCF, TPO, FL, and IL-3 and found that this combination could expand human CB CD133-sorted SRCs by 5.8-fold in a serum- and stromal cell-free condition.5 K7 }/ I& r8 H4 A/ l

MATERIALS AND METHODS

Separation of CD133- and CD34-Enriched Cells from Human CB
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Human CB samples were collected from normal full-term deliveries after informed consent was obtained. Mononuclear cells (MNCs) were separated by density gradient centrifugation (Lymphoprep; Axis-shield, Oslo, Norway, http://www.axis-shield.com) after depletion of phagocytes with Silica (Immuno-Biological Laboratories Co., Takasaki, Gunma, Japan, http://www.ibl-japan.co.jp). CD133- and CD34-enriched cells were separated from MNCs by using magnetic cell sorting (MACS) CD133 MicroBead Kit or MACS Direct CD34 Progenitor Cell Isolation Kit (hereafter CD133-MACS and CD34-MACS, respectively; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), respectively. In some experiments, separated cells were examined by flow-cytometric analyses using FcR Blocking Reagent, fluorescein isothiocyanate (FITC)-conjugated anti-human CD34, allophycocyanin (APC)-conjugated anti-human CD133 (clone 293C3) (Miltenyi Biotec), phycoerythrin (PE)-conjugated anti-human CD38 antibodies (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), and 7-amino-actinomycin D (7-AAD) (Via-Probe; BD Pharmingen). The yield of the target cells was calculated as follows:  x 100 (%).! Q* |* T' `' _' B6 g+ C

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Recombinant human SCF, TPO, IL-3, and IL-6/sIL-6R chimeric protein FP6 were generated by Kirin Brewery Co., Ltd. (Tokyo, http://www.kirin.co.jp/english), and the recombinant Delta1-Fc chimeric protein was generated as previously described . These reagents were certified as free from endotoxin (- T* `- O4 }% g! t) F$ f
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Cell Culture1 O. H+ D0 F  e! n+ m

Plates or dishes not treated with tissue culture were precoated with 10 µg/ml Delta1-Fc or control Fc fragment of human IgG (IgG-Fc) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) followed by 10 µg/ml human fibronectin (Boehringer Ingelheim GmbH, Ingelheim, Germany, http://www.boehringer-ingelheim.de). Cells were cultured in serum-free medium composed of Iscove's modified Dulbecco's medium (IMDM) supplemented with 10 mg/ml bovine serum albumin, 10 µg/ml human insulin, 200 µg/ml human transferrin, 2 mM L-glutamine, 0.1 mM 2-Mercaptoethanol, 4.7 µg/ml linoleic acid, 4.7 µg/ml oleic acid, and 8 µg/ml cholesterol (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, http://www.kyokutoseiyaku.co.jp) at 37¡ãC in a humidified atmosphere flushed with 5% CO2 in air. Cytokines were added at concentrations of 100 ng/ml for SCF, 10 ng/ml for TPO, 100 ng/ml for FL, 100 ng/ml for FP6, 100 ng/ml for IL-6, and 10 ng/ml for IL-3. Cell culture was initiated in 24-well plates and serially transferred to six-well plates and 10-cm dishes to avoid overgrowth of the cells. Half of the culture medium was changed every 2 or 3 days.; h* |% ^( p0 ~# X" `! R! R

Colony Assays7 z  P3 D/ R5 E7 @

At the indicated time points, cultured cells were harvested and plated in a semisolid medium, Methocult GF H4434, containing IMDM with 30% fetal bovine serum (FBS), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml human SCF, 10 ng/ml human granulocyte-macrophage colony stimulating factor, 10 ng/ml human IL-3, and 3 units/ml human erythropoietin (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) and incubated at 37¡ãC. Colony-forming ability was assessed after 15¨C16 days of culture.

Transplantation to Nonobese Diabetic/SCID or Nonobese Diabetic/SCID/cnull Mice

To assess the in vivo repopulating capacity of isolated cells and their cultured progeny, we used nonobese diabetic (NOD)/SCID (NOD/Shi-scid; CLEA Japan, Inc., Tokyo, http://www.clea-japan.com) and NOD/SCID/cnull (NOG) mice . Mice were fed with autoclaved acidified water and sterilized food. At 10¨C13 weeks after transplantation, mice were sacrificed, and cells were harvested from both femurs, peripheral blood, spleen, and thymus. In the indicated experiments, analyses were performed 24 weeks after transplantation. In the limiting dilution transplantation analyses, we transplanted cells into six to 12 recipient mice in each limiting dose for reliable estimation.
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In the serial transplantation experiment, we isolated bone marrow cells from the primary NOG recipient mouse 24 weeks after the first transplantation, and MNCs were separated by density gradient centrifugation (Histopaque-1083; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). The MNCs were divided into three aliquots and injected intravenously into secondary NOG recipients. Ten weeks after the second transplantation, bone marrow cells were harvested and analyzed.  Z9 j/ ]* q& h) z, L* b

Flow-Cytometric Analysis of Transplanted NOD/SCID and NOG Mice4 P- g0 \: M$ {2 q9 S. K; e) v
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Engraftment of human cells was examined by analyzing human surface antigens using BD LSR2 (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Cells harvested from the bone marrow, peripheral blood, spleen, and thymus of recipient mice were treated with ammonium chloride red blood cell lysis buffer (Sigma-Aldrich) and blocked with PBS containing 2% FBS, anti-mouse CD16/32 antibody (BD Pharmingen), and FcR Blocking Reagent (Miltenyi Biotec). Then, they were stained with FITC-conjugated anti-human CD45 (clone HI30; BD Pharmingen) and anti-human CD3 (Beckman Coulter, Inc., Fullerton, CA, http://www.beckmancoulter.com), PE-conjugated anti-human CD13, CD33, CD56, CD4 (Beckman Coulter, Inc.), and CD133 (clone 293C3; Miltenyi Biotec), APC-conjugated anti-murine CD45 (clone 30-F11; BD Pharmingen), anti-human CD3, CD19, CD8 (Beckman Coulter, Inc.), and CD34 (Miltenyi Biotec), and 7-AAD (Via-Probe; BD Pharmingen). Successful engraftment of human hematopoietic cells was determined by detection of greater than 0.1% of human CD45  cells in recipient bone marrow cells.2 M% D- E( f, Y9 [8 I! m0 d
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Limiting Dilution Analysis

The frequencies of SRCs capable of repopulating in NOD/SCID mice were quantified by a limiting dilution analysis by applying Poisson statistics to the single-hit model as described previously . The frequencies of SRCs and statistical comparison between individual populations were calculated by using L-Calc software (StemCell Technologies).8 Z6 B- Z8 o- }

Statistical Analysis1 \- X' d4 q! Y. t
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Data are presented as mean ¡À SEM. Analysis of statistical significance was determined by paired t test., h& Y3 U% S8 e! t- q9 V" {4 ^

RESULTS
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Stem Cell Isolation by the CD133-MACS Recovers a Higher Number of SRCs than by CD34

Because several investigators have suggested that SRCs are more concentrated in CD133  cells than in CD34  cells , we directly compared the frequency of SRCs contained in the populations sorted by CD133-MACS and CD34-MACS. Flow-cytometric analyses of four CB samples showed that 0.2%¨C1.4% (mean 0.8%) and 0.8%¨C3.0% (mean 1.9%) of MNCs were positive for CD133 and CD34, respectively. More than 98% of CD133  cells were CD34 , and approximately 43% (25%¨C56%) of CD34  cells were CD133  (Fig. 1A, a).

Figure 1. Separation of CD133- or CD34-enriched cells from CB MNCs and comparison of their in vivo repopulating capacity. (A): Expression profiles of CD133 and CD34 on CB MNCs (a) and cells separated by CD133- (b) and CD34-MACS (c). Representative data among several samples are shown. (B): The repopulating ability of CD133- and CD34-sorted cells isolated from the same CB samples (1 and 2). Frequencies of SRCs estimated by limiting dilution analyses are shown. The lower panels show chimeric proportion of human CD45  cells in the bone marrow of recipient mice, and the number of transplanted mice is shown in the upper-left margin of the panels. (C): Estimated frequencies and numbers of SRCs in the transplanted samples. In both experiments, CD133-sorted cells contain higher frequencies of SRCs than CD34-sorted cells, and CD133 sorting provides higher numbers of SRCs from the same volume of original MNCs or NCs than CD34 sorting. Abbreviations: CB, cord blood; MACS, magnetic cell sorting; MNC, mononuclear cell; NC, nucleated cell; SRC, severe combined immunodeficient repopulating cell.
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Then, we prepared two identical CB MNC aliquots and isolated CD133- and CD34-enriched cells by CD133- and CD34-MACS. Flow-cytometric analyses after isolation showed that the purities of separated cells were variable among samples (53.1%¨C93.5% for CD133 and 53.9%¨C96.3% for CD34), but there was no significant difference between the two separation methods (p = .12). Calculated recovery rates of the target cells (see Materials and Methods) were 66% ¡À 10% for CD133 and 46% ¡À 10% for CD34, showing a tendency of better recovery of CD133 cells by CD133-MACS than recovery of CD34 cells by CD34-MACS, but the difference was not significant (p = .06). After separation, approximately 75% of the CD34-sorted cells were CD133 , whereas virtually all of the CD133-sorted cells were CD34  with only rare (0.1%) CD34¨C cells in most of the samples (Fig. 1A, b and c). Based on the comprehensive calculation, the recovery rates of CD133 CD34  cells (i.e., the major SRC-containing population) in the individual samples were 66% ¡À 10% and 83% ¡À 8% by CD133- and CD34-MACS separation, respectively. The recovery efficiency of this most immature fraction by CD34-MACS tended to be superior to the one by CD133-MACS, but again it was not significantly different (p = .14).
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Then, to compare the number of SRCs contained in the populations separated by CD133- and CD34-MACS, we transplanted cells of each population into irradiated NOD/SCID mice intravenously and examined their in vivo hematopoietic repopulating capacity. To evaluate the number of SRCs quantitatively, we transplanted serially reduced numbers of cells. Frequencies of SRCs in the CD133-sorted population were one of 1,454 and 947 in samples 1 and 2, respectively. In contrast, those in the CD34-sorted population were one of 9,306 and 5,904 in samples 1 and 2, respectively (Fig. 1B, 1C). This means that SRCs were sixfold more concentrated in the CD133-sorted population than in the CD34-sorted one. Converting this frequency into the absolute number of SRCs obtained from the same number of primary MNCs, CD133 sorting recovered 308 (sample 1) and 254 (sample 2) SRCs, and CD34 sorting recovered 67 (sample 1) and 59 (sample 2) SRCs from 108 of total MNCs (Fig. 1C). Therefore, despite the similar recovery rate of CD133 CD34  cells by CD133- and CD34-sorting procedures, CD133 sorting provides 4.3¨C4.6-fold greater absolute numbers of SRCs than CD34 sorting. Thus, for our subsequent SRC expansion experiments, we used the CD133-sorted population as the culture-initiating cells., e0 M, \" e2 a3 G6 O

Immobilized Delta1-Fc Chimeric Protein Can Expand Immature CB Hematopoietic Precursors in the Presence of Cytokines

Before evaluating methods for HSC expansion ex vivo, we first explored optimal culture conditions to expand immature hematopoietic precursors by using various combinations of hematopoietic cytokines and soluble Notch ligands. Because immobilization of Notch ligands has been demonstrated to be important for their efficient activity .4 g! H9 ?/ Y3 \( E2 t# g

We cultured CB CD133-sorted cells in four cytokine combinations of (a) 3GFs IL-6, (b) 3GFs IL-6 IL-3, (c) 3GFs FP6, and (d) 3GFs FP6 IL-3, plus additional conditions with Delta1-Fc or IgG-Fc in each cytokine combination, and compared the expansion rate of total cells, CD133 CD34 CD38¨C immature hematopoietic cells, and mixed colony-forming cells (CFU-Mix). All culture conditions increased the number of total cells and CD133 CD34 CD38¨C cells during 3-week culture (Fig. 2A, 2B). Addition of IL-3 or replacement of IL-6 with FP6 gave greater expansion of total cells and CD133 CD34 CD38¨C cells. However, Delta1-Fc had very little effect on the expansion of these cells (Fig. 2A, 2B).2 c/ u0 m/ O) N5 R" ~4 r. p. x: I
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Figure 2. The expansion rates of total cells, CD133 CD34 CD38¨C immature cells, and CFU-Mix. CB CD133-sorted cells were cultured in the presence of indicated cytokines and Notch ligands for 1, 2, and 3 weeks. (A, B): The numbers of total cells (A) and CD133 CD34 CD38¨C immature hematopoietic cells (B) were counted, and the expansion rates are shown. (C): After indicated periods of culture, cells were replated in a semisolid medium and the number of CFU-Mix was evaluated. The expansion rate of the number of CFU-Mix is shown. Abbreviations: 3GFs, three growth factors; CFU-Mix, mixed colony-forming cells; D1, Delta1-Fc; Fc, IgG-Fc; FP6, interleukin-6/soluble interleukin-6 receptor chimeric protein; IL, interleukin; wk, week./ F9 w  n6 R1 s8 ^! A: ]$ r2 e8 r- L
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In contrast, addition of IL-3 was always required for the consistent expansion of CFU-Mix until 3 weeks (Fig. 2C). In the presence of IL-3, addition of FP6 increased the number of CFU-Mix significantly better than IL-6 (p

Effects of IL-6-gp130, IL-3, and Notch Signalings on SRC Expansion in the Serum-Free Culture

We have found that the number of CFU-Mix was continuously increased until 3 weeks in several conditions (Fig. 2C) and declined thereafter (data not shown). And although a previous report demonstrated that serum-containing culture with 3GFs and IL-6/sIL-6R for 1 week increased the number of SRCs by fourfold, no increase of human blood cell chimerism in recipient mice was observed when we cultured cells for 1 week in the serum-free conditions with either 3GFs FP6 or 3GFs FP6 IL-3 Delta1-Fc (data not shown). Based on these observations, we determined to culture cells for 3 weeks to evaluate SRC expansion.

As shown in Figure 3A, all the mice transplanted with more than 5,000 fresh CD133-sorted cells showed engraftment, but fewer than 2,500 cells failed to engraft in some of the mice. The frequency of SRCs in this sample was calculated as one of 1,020 (95% confidence interval .

Figure 3. The repopulating ability of fresh CB CD133-sorted cells and their progenies after the culture with various combinations of cytokines for 3 weeks. (A¨CE): The frequencies of SRCs in fresh CD133-sorted cells (A), cells cultured with SCF TPO FL (3GFs) FP6 (B), 3GFs FP6 Delta1-Fc (C), 3GFs FP6 IL-3 Delta1-Fc (D), and 3GFs IL-6 IL-3 Delta1-Fc (E). They were estimated as 1/1,020 (A), 1/640 (B), 1/361 (C), 1/175 (D), and 1/266 (E), respectively, by limiting dilution analyses. The right panels show chimeric proportion of human CD45  cells in the bone marrow of recipient mice, and the number of transplanted mice is shown in the upper-left margin of the panels. (F): Integrated representation of (A¨CE). Correspondence of the symbols and lines is noted in the right. Abbreviations: 3GFs, three growth factors; FL, flt-3 ligand; FP6, interleukin-6/soluble interleukin-6 receptor chimeric protein; IL, interleukin; SCF, stem cell factor; TPO, thrombopoietin.; K7 N' n+ U5 m9 Q# `8 s
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In contrast, when immobilized Delta1-Fc was present in the same cytokine combination (i.e., 3GFs FP6), the frequency of SRCs increased to the equivalent to one of 361 (95% CI, 1/218¨C1/596) culture-initiating cells, indicating 2.8-fold SRC expansion compared with the SRC number before culture (p = .005, Fig. 3C). The addition of IL-3 to this condition further augmented the expansion efficiency, achieving the SRC frequency of equivalent to one of 175 (95% CI, 1/109¨C1/279) culture-initiating cells, indicating 5.8-fold expansion (p = .0001, Fig. 3D). To our knowledge, this ranks with the highest human SRC expansion efficiency ever reported. It is of note that two of six mice transplanted with cultured progeny equivalent to 60 culture-initiating cells showed human blood cell chimerism. To further compare the effects of IL-6 and FP6, we replaced FP6 with IL-6. In this condition, SRC frequency was equivalent to one of 266 (95% CI, 1/159¨C1/446) culture-initiating cells. The expansion rate was reduced from 5.8-fold to 3.8-fold, although significant expansion was still achieved (p = .0006; Fig. 3E).

Taken together, significant SRC expansion was realized in all three conditions with immobilized Delta1-Fc chimeric protein. Among these, combination of Delta1-Fc, IL-3, and IL-6/sIL-6R chimeric protein, FP6, in addition to 3GFs, provided the most significant expansion in the serum-free condition. It is noteworthy that IL-3 showed a positive effect in this condition, in contrast to the negative impact in the serum-containing condition without Notch signaling .
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SRCs Cultured for 3 Weeks in the Serum- and Stromal Cell-Free Condition with 3GFs, FP6, IL-3, and Delta1-Fc Normally Contribute to Myeloid, B, T, and NK Cell Lineages in NOG Recipient Mice and Repopulate Recipients of Secondary Transplantation
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To examine the long-term in vivo myeloid and lymphoid repopulating capacity of the cells cultured with 3GFs, FP6, IL-3, and Delta1-Fc, we transplanted these cells into NOG mice, which were generated by intercrossing NOD/SCID mice with IL-2 receptor common  chain-knockout (cnull) mice. These mice, unlike NOD/SCID mice, are known to allow transplanted human HSCs/HPCs to differentiate even into the T-cell lineage , and therefore we could examine the in vivo differentiation capacity of the ex vivo expanded HSCs most efficiently. These mice also have the advantage of higher engraftment of transplanted human cells. We cultured 10,000 CB CD133-sorted cells for 3 weeks and transplanted them into NOG mice. After 12 weeks, we observed 53%¨C67% human CD45  cells in the recipient bone marrow. Further analyses of the bone marrow, peripheral blood, spleen, and thymus of recipient mice revealed that human hematopoietic cells differentiated into myeloid (CD13  or CD33 ), B (CD19 ), T (CD3 ), and NK (CD56 ) cell lineages (Fig. 4A). In addition, in the bone marrow of recipient mice, we detected CD133 CD34  immature hematopoietic cells at frequencies of 0.5%¨C1.1% of human cells. In the thymus, human cells represented virtually all the CD3  cells (data not shown), and among the CD3  cells, the patterns of differentiation to CD4/CD8 double-positive, CD4 single-positive, and CD8 single-positive cells were very similar to that of normal thymocytes (Fig. 4A). Robust human hematopoietic repopulation was confirmed in another recipient mouse 24 weeks after transplantation (Fig. 4B). In this mouse, more definite reconstitution of CD3  mature T cells was observed in the peripheral blood and spleen.
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Figure 4. In vivo repopulating and differentiation capacity of the cells cultured with 3GFs FP6 IL-3 Delta1-Fc. Cord blood CD133-sorted cells were cultured with 3GFs FP6 IL-3 Delta1-Fc for 3 weeks and were transplanted into NOG mice. The BM, PB, SP, and TH of recipient mice were collected 12 weeks (A) and 24 weeks (B) after transplantation, and contribution of human cells to various hematopoietic lineages was examined by flow-cytometric analyses. (A): Representative data of recipient mice examined 12 weeks after transplantation. Human CD45  cells accounted for 53.5% of total BM cells (a), and a substantial number of human CD3  (b, f), CD13  (b), CD33  (b), CD19  (c, e), and CD56  (c, e) cells were detected in the BM and spleen. CD133 CD34  immature hematopoietic cells were also clearly identified (1.1%) in the BM (d). In the thymus, CD3  cells expressed CD4 and/or CD8 (g) showing a solid development of human T cells. (b¨Cg) represent data gated by human CD45  cells. (B): Flow-cytometric data from a mouse examined 24 weeks after transplantation. A high level of engraftment (a) (39%), reconstitution of CD133 CD34  immature cells (d) (2.1%), and contribution to myeloid (b), B-cell (c, e, f, h), T-cell (b, f¨Ci) and NK-cell (c, e) lineages were confirmed in the BM, spleen, thymus, and peripheral blood. (b¨Ci) represent data gated by human CD45  cells. Abbreviations: 3GFs, three growth factors; BM, bone marrow; FP6, interleukin-6/soluble interleukin-6 receptor chimeric protein; IL, interleukin; NK, natural killer; PB, peripheral blood; SP, spleen; TH, thymus.
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To confirm that transplanted HSCs still retain their self-renewal capacity after primary transplantation, we collected bone marrow cells 24 weeks after the transplantation from a primary recipient, which had been transplanted with the progeny of 1 x 104 CB CD133-sorted cells ex vivo expanded, and injected them into three secondary NOG mice. Ten weeks after the secondary transplantation, we observed engraftment of human CD45  cells (0.1%) in the bone marrow of two recipient mice (Fig. 5A), and human hematopoietic cells differentiated into myeloid (CD13  or CD33 ) and lymphoid (CD19 ) cells (Fig. 5B). These findings strongly indicate that cells cultured with 3GFs, FP6, IL-3, and Delta1-Fc for 3 weeks retain long-term repopulating capacity and normal differentiation capacity in vivo.& O7 ?- l% J, v) r3 m: q

Figure 5. Cells cultured with three growth factors (3GFs) interleukin (IL)-6/soluble receptor chimeric protein (FP6) IL-3 Delta1-Fc retain long-term repopulating capacity after serial transplantation into secondary nonobese diabetic/severe combined immunodeficient/cnull (NOG) recipients. Ten-thousand cord blood CD133-sorted cells were cultured with 3GFs FP6 IL-3 Delta1-Fc for 3 weeks and were transplanted into a primary NOG mouse. Twenty-four weeks after transplantation, bone marrow (BM) cells were harvested and serially transplanted into three secondary NOG recipients. (A): Ten weeks after secondary transplantation, BM cells of recipient mice were harvested and chimerism of human cells was analyzed. Two of the three secondary recipients showed substantial human engraftment. (B): Representative flow-cytometric data of BM cells in a secondary recipient (mouse S1). Human myeloid (CD13  or CD33 ) and lymphoid (CD19 ) cells can be identified. Data with isotype controls are shown as insets in the upper-left margin of the figures.2 O9 P' @, Q" N* Z0 W" x* A
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DISCUSSION  N5 j: A% q/ o* @* c9 U% D: z; c& o
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Efficient Ex Vivo Expansion of SRCs
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In this study, we demonstrated successful expansion of SRCs by approximately sixfold, by culturing human CB CD133-enriched cells with SCF, TPO, FL, FP6, IL-3, and Delta1-Fc. SRCs have now been widely accepted as the most immature human hematopoietic cells and are regarded as surrogates for HSCs . Our analysis, satisfying these criteria, revealed expansion of SRCs, which ranks as the most efficient one. Our method also enabled a 240-fold expansion of CFU-Mix, demonstrating its surprisingly strong effect on expanding immature progenitors.+ C) i/ o7 J2 N4 P4 k
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We also demonstrated that the cultured cells can differentiate in vivo into myeloid, B, T, and NK cell lineages in the bone marrow, peripheral blood, spleen, and thymus in NOG mice. Human cells transplanted into NOG mice can engraft at significantly higher levels than NOD/SCID mice, and transplanted cells can differentiate even to the T-cell lineage. Based on these features, NOG mice are increasingly used as recipients of human stem cells as well as NOD/SCID/ß2-microglobulin null mice . We found immature CD133 CD34  human cells in the bone marrow of recipient NOG mice at a substantial frequency, and after serial transplantation, progeny of the cultured cells engrafted most of the secondary recipients. These findings suggest that the culture system preserves normal stem cell functions.

Positive Effects of Notch Signaling on SRC Expansion8 ?8 k: L' N" E1 f
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A positive effect of soluble Notch ligands on human SRCs was previously suggested by two groups, although they did not confirm the increase of SRCs quantitatively . It is thus interesting to combine our system with hypoxic conditions for further better efficiency of ex vivo HSC expansion./ h+ K% u& F% W1 F

Effects of IL-3 and gp130 Signaling Pathways on SRC Expansion

We found that IL-3 exerts positive effects on amplifying SRCs at least in the presence of SCF, TPO, FL, FP6, and Delta1-Fc in a serum-free condition. To date, many researchers have examined the effects of IL-3 on HSCs, but the results have been controversial: some reports showed maintenance of HSCs, whereas others showed negative effects . This discrepancy may depend on the addition of serum, the difference of coexisting cytokines, and the culture-initiating cells. Our result may suggest that IL-3 has additive or synergistic effects with Delta1-Fc on HSCs in the absence of serum.

We also found that replacement of IL-6 with FP6 had some superior effects on SRC expansion. Unlike the addition of IL-3, however, the effects of FP6 were marginal in the presence of Delta1-Fc and IL-3. This could be because the combination of Delta1-Fc and IL-3 could transmit nearly optimal growth signals in HSCs. Or the difference of the cell source (i.e., CD133- vs. CD34-sorted cells) might explain the results .

Stem Cell Source for Transplantation and Ex Vivo Culture" v. g+ S' G- d" @$ m

To obtain the maximum efficiency of stem cell expansion, the isolation method for culture-initiating cells is also very important. CD34 sorting has been most widely used for positive selection of HSCs in the clinical practice. Recently, feasibility of the CD133-sorted cell transplantation has been evaluated in several clinical trials , and this biological property may have reduced the SCID repopulating capacity of the CD34-sorted cells.

Recently, a small population of CD133 CD34¨C(Lineage¨C . Therefore, although these cells account for no more than 0.1% of the CD133-sorted cells, the use of CD133-sorted cells as the culture-initiating cells may help increase the absolute number of HSCs after culture.

All these considerations imply that CD133-sorted cells are more advantageous as a direct source for HSC transplantation and as a culture-initiating source for ex vivo HSC expansion than CD34-sorted cells to obtain a greater number of HSCs. According to our results, we can estimate that culturing CD133-sorted cells with Delta1-Fc yields as many as approximately 25-fold greater numbers of HSCs compared with the fresh CD34-sorted cells. Currently, clinical devices for CD34 sorting which use QBend10 or other Class II anti-CD34 antibodies are widely used. Our findings suggest that CD133 sorting might be a better way to collect and enrich HSCs than CD34 sorting by QBend10 or other ClassII antibodies. Future studies that directly compare the clinical outcome of CD133 and CD34 sortings may deepen our understandings for effective enrichment of HSCs.0 E) ~' A, A( I& D$ k
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CONCLUSION

In this report, we have demonstrated that serum-free culture of the CD133-sorted human CB cells in the presence of SCF, TPO, FL, FP6, IL-3, and Delta1-Fc is an optimized condition to obtain the highest number of ex vivo expanded HSCs. Based on this condition, some additional explorations should be considered. Increase of differentiated cells surpasses that of immature cells, which might interfere with the SRC expansion because the differentiated cells might secrete various substances that inhibit SRC expansion. Indeed, removal of differentiated cells during culture of CB Lin¨C cells has been shown to have strongly positive effect on the efficient SRC expansion in a serum-free culture with 3GFs . Therefore, a much higher level of SRC expansion might be possible if we apply similar differentiated cell-removal protocols in our culture condition. Culture under the hypoxic condition may also improve the expansion efficiency. In this study, we expanded HSCs by a 3-week culture system. In general, shorter ex vivo culture periods are preferable in clinical settings from the viewpoint of safety or costs. Further studies based on our results and above ideas may provide improved methods with shorter culture periods and higher expansion efficiency, which could be the most efficient ex vivo HSC expansion system for clinical applications in the future.2 O3 o4 g7 Y  M5 n: Y1 R$ h
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DISCLOSURES; ~) [' v# v% M

M.N. owns stock in and has received financial support from Kirin Brewery Co., Ltd. S.S. owns stock in and has received financial support from Asahi Kasei Corporation.  r) O. Z& E$ @( d! ~; s
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ACKNOWLEDGMENTS

This work was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Research on Pharmaceutical and Medical Safety, Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan and Grant-in-Aid for Scientific Research, KAKENHI (17014023) from the Japan Society for the Promotion of Science, and a grant from the Takeda Science Founda-tion. We thank Dr. A. Kikuchi for the supply of CB samples and Kyokuto Pharmaceutical Industrial Co., Ltd. for generously providing us with the serum-free medium. We also thank Y. Sato for taking care of the animals.) ?: L- S. }3 P2 n6 E4 Q) n
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