标题: Simple and Efficient Isolation of Hematopoietic Stem Cells from H2K-zFP Transgen [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:47 标题: Simple and Efficient Isolation of Hematopoietic Stem Cells from H2K-zFP Transgen
a Department of Life Science, Swiss Federal Institute of Technology, Lausanne, Switzerland;: o, g% N( ~8 t2 g( |8 w8 [( }
7 j p5 X0 h- f; F! r& E \b The Burnham Institute, La Jolla, California, USA; 9 _: Z, O# D( M' y0 w7 q6 U0 |' L! q. t' f
c Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California, USA , O# M4 m# F3 Z. w. h" E7 x! L1 Y9 ` X1 w
Key Words. Hematopoietic stem cells ? Fluorescent protein ? Prospective isolation 7 S% X- g1 S" W; G+ B! v1 t1 i) q6 X8 R8 _- S; N; R
Correspondence: Alexey V. Terskikh, Assistant Professor, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA. Telephone: 858-646-3100 (ext. 3624); Fax: 858-713-6274; e-mail: Terskikh@Burnham.org5 A; d- ~& U, z; Z- f: [
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ABSTRACT 7 E6 y$ E# K. [) s3 x6 H- b+ k6 A8 P: ?* [$ n
The therapeutic potentials of somatic stem cells have been widely recognized. To harness the power of stem cells, however, the molecular mechanism underlying self-renewal and differentiation must be uncovered. Most current experimental strategies aimed at understanding stem cell homing to stem cell niches as well as self-renewal and differentiation in vivo and in vitro include gene overexpression or knockout/knockdown in stem cells. For instance, the importance of genes such as p21 , p27 , SCL , Bcl2 , Bmi1 , Bmpr1 , and many others was demonstrated using the knockout or transgenic approach. Such experiments can greatly benefit from a simple technique to prospectively isolate a cell population highly enriched in stem cells.- g- g9 v7 Q" C" b9 l' B
" C& {; c( j" s& r P$ fIn the field of hematopoiesis, the prospective isolation of hematopoietic stem cells (HSCs) from adult mouse bone marrow , followed by prospective isolation of lymphoid and myeloid progenitor populations , allowed the gene expression analysis of these highly purified, functionally homogeneous cell populations . In the case of mouse HSCs, the combination of c-kithigh, Sca-1high, and Linneg markers defines the stem cell pool, which can be further subdivided using Mac-1, CD4, Thy-1.1, or Flk2 markers . More recently, long-term HSCs were isolated using a slightly different combination of markers, namely EndohighSca-1highLinneg/loworEndohighSca-1highRholow (Rhodamine-123 low) . In both cases, the isolation of stem cells by flow cytometry requires a complex cocktail of multicolor fluorescence-labeled monoclonal antibodies〞approximately 7–10 monoclonal antibodies (mAbs) with at least four distinct fluorochromes. This setup is demanding in terms of cost and time, requires a multilaser fluorescence-activated cell sorter (FACS) machine, and remains a state-of-the art procedure rather than a routine cell sorting, which puts the isolation of pure HSCs out of reach for many excellent laboratories not specialized in the HSC field. + ?0 P0 y6 F" I. y" x- C) _; c: U. O, ^* d' `2 l3 `; b
More than 20 years ago, Visser et al. noted that the H2K determinant is highly expressed in the HSC compartment and thus can be used to enrich HSCs. We took advantage of these exceptionally high levels of gene expression from the 2kB H2Kb promoter/enhancer element in long-term HSCs. In this study, we describe a simple way to isolate a cell population that is highly enriched in HSCs from adult mouse bone marrow using a transgenic mouse strain that expresses a green fluorescent protein (GFP) variant (zFP) under the control of the H2Kb promoter.7 ]% ]: r7 n& {* b6 j, N6 S
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RESULTS % `( L3 k: E' F) p: U 7 ^3 |0 A9 ^# o* W! ZThe major objective of this work is to facilitate the critical enrichment of a very rare population of HSCs and to make them readily available in any laboratory as a simple routine rather than a sophisticated procedure that requires considerable expertise in flow cytometry and multicolor sorting capabilities.0 d9 k( ? ?- n' h0 r7 P5 } Q
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We have described a simple way to isolate highly enriched genetically labeled HSCs from the enriched bone marrow of H2K-zFP transgenic mice using a built-in zFP fluorescent reporter. We have provided strong evidence that most (60%–90%) zFPbright cells from the c-kit–enriched bone marrow of H2K-zFP transgenics are phenotypically and functionally identical to the previously characterized c-kithigh, Sca-1high, Linneg, Thy-1.1low/Flk-2neg long-term HSCs . As opposed to conventional methods, our protocol does not require any fluorescent-conjugated antibody and is based on a simple one-step enrichment procedure, which is a routine used by any method aimed at prospective isolation of significant numbers of HSCs. The enrichment step is flexible, because both c-kit and Sca-1 antigens found to be highly expressed on HSCs can be used. At present, the availability of anti–Sca-1 directly conjugated MicroBeads (Miltenyi Biotec, Auburn, CA, http://www.miltenyibiotec.com) makes this one-step enrichment into a simple "out-of-the-box" procedure.! R5 f+ }* o3 i1 J& Q
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We have used in vivo reconstitution analysis, the gold standard, to determine the potency of zFPbright cells as HSCs. A combined judgment from the surface phenotype and in vivo reconstitution assays would suggest that zFPbright cells on average contain from 70%–80% long-term HSCs. The size of the stem cell compartment (determined to be ~10 cells in the limiting dilution analysis) in the whole bone marrow of C57BL/6 mice is approximately 0.01% of total bone marrow cells . In our hands, the c-kit enrichment procedure reproducibly results in an approximately 20-fold enrichment of the stem cell population. Thus, the numbers of zFPbright cells (i.e., 0.2% representing the brightest zFP cells) that we empirically found to be highly enriched in HSCs are comparable to the HSC population in the bone marrow.$ o& q" ~0 l* D1 w
$ F& H8 ^1 c/ `2 _& ]- w" T9 eA method to enrich for pluripotent HSCs described by Visser et al. consisted of three separation steps that use density-gradient and wheat-germ agglutinin-FITC conjugate enrichment followed by H2K-biotin avidin-FITC labeling and isolation of cells with high H2K density . Although more laborious, the principle of this procedure is similar to the c-kit or Sca-1 enrichments of whole bone marrow followed by the isolation of zFPbright cells. Using the spleen colony assay, Visser et al. estimated the purity of putative HSCs to be on average 65% (6.6 colonies per spleen on day 12 spleen-seeding efficiency factor = 0.1), although this seems to be the readout for multipotent myeloid progenitors rather than pluripotent long-term HSCs. The absence of a limiting dilution assay in the earlier studies complicates the functional comparison of the two approaches. However, the bright cells detected with the H2kb antibody and the zFPbright cells constitute overlapping but not identical populations (Fig. 3, bone marrow). - u& l% j* l& n: f$ l4 i; i9 \9 s# G ]6 E: }* x/ v- u0 d5 u' D
The cells in SP population, originally described by Goodell et al. , are enriched in HSC activity. We found that approximately 37% of all zFPbright cells in enriched H2K-zFP bone marrow are contained within SP and the mean zFP fluorescence of SP cells is more than 10-fold higher than average. On the other hand, approximately 16% of SP cells are zFPbright, suggesting a functional overlap between zFPbright and SP cells. Recently, Matsuzaki et al. combined the SP strategy and conventional HSC labeling as described above to purify a subset of the long-term HSCs. A highly efficient homing and reconstitution capacity was reported for the cells contained within the very tip of the SP: Tip-SP, CD34–, c-Kit , Sca-1 , Lin– cells (Tip-SP/CD34–/KSL). We note that mean zFP fluorescence is almost two times higher in SP tail (similar to Tip-SP) than in SP top population and the brightness of zFP cells correlates with their enrichment in the SP tail fraction. However, even among 0.02% brightest zFP cells, approximately half of these cells are found outside of SP fraction, which suggests that some HSCs are found outside of the SP fraction. On the other hand, zFPbright cells are almost entirely (~96%) CD34-negative, in agreement with previous findings that most primitive HSCs lack the CD34 marker . These results indicate that a combination of zFPbright phenotype with Hoechst staining may allow isolation of HSCs having exceptionally high homing activity. We are currently investigating whether the combination of Tip-SP/zFPbright parameters will yield an engraftment efficiency similar to that previously described for the Tip-SP/CD34–/KSL combination. Alternatively, it will be of interest to investigate if the zFPbright cells outside of SP are true HSCs. + G) x+ V# s X5 M2 { * m: x* w* ?4 l/ e7 `( T; j+ E( APreviously, several groups used an enhanced GFP (EGFP) marker under the control of the Sca-1 promoter to enrich for HSC/ progenitor populations . However, a relatively modest enrichment of approximately 100-fold (meaning approximately 1 HSC in 100 cells) was achieved in one case and a minimum of only 750 GFP cells were tested in the other case . However, the report did not describe the limiting dilution capacity of the Sca-1 EGFP-enriched cells. Thus, our limiting dilution analysis suggests at least a 10-fold improvement in the long-term HSC enrichment in zFPbright population compared with previous reports .1 J* u( w9 e# q4 v
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The empiric definition of zFPbright cells as the top 0.2% of zFP-positive cells may seem vulnerable at first sight. However, in practice this simple rule performed very well in several reconstitution experiments consistently yielding the reconstitution levels reported in this paper. Indicative of this robustness is our serendipitous observation of a correlation between zFP and Sca-1 expression. Despite the arbitrary selection of zFP expression gates, well-discernable populations of c-kit–positive cells with variable Sca-1 expression level can be observed. We speculate that this population represents a mixture of various short-lived progenitor populations. If this hypothesis is correct, one would be able to simultaneously isolate the short-term progenitor and long-term HSC populations from the same sample of the c-kit–enriched bone marrow of H2K-zFP mice. This would make H2K-zFP transgenics a useful tool to address questions related to the mechanisms of HSC self-renewal versus the commitment of more short-lived progenitors. . y& I4 S e9 Z% L$ M 3 ~$ P A! A$ b/ O6 |3 [, c. Y% tAlthough the zFPbright population is highly enriched for HSCs, other cells, including various committed hematopoietic progenitors as well as mesenchymal cells, might be present in zFPbright population in addition to HSCs. Nevertheless, this strategy represents a major improvement over the previous promoter-reporter combinations and over a single marker (i.e., c-kit or Sca-1) enrichment, which is often used by many researchers for gene transfer and transplantation experiments. A typical example of zFPbr ig ht population usage would be for experiments aimed at investigating gene expression effects on the HSC compartment or the entire bone marrow. For instance, the lentiviral-mediated gene transfer (including lentiviral short interfering RNA for the purpose of RNA interference) into zFPbright cells followed by the transplantation into lethally irradiated C57BL/6 is a straightforward and simple experiment. High enrichment in HSCs is particularly useful when the viral titers are low or the infection is inefficient, which is inevitable for large cDNAs. The intrinsic green fluorescence of H2K-zFP transgenic cells will encourage the use of a variety of fluorescent reporter proteins , especially the red fluorescent proteins DsRed, fluorescent timer, fast DsRed, monomeric DsRed, and the far-red fluorescent protein to visualize the cells transformed with the desired vector.6 F& ^. x, v' e
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Experimental Protocols( k0 |; W0 U5 Z" M. u6 b2 W
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Plasmids ? The H2K-i-LTR cassette consisting of the H2Kb-promoter/ enhancer element H2Kb intron sequence and Moloney MuLV enhancer/poly(A) site was described . We used the NotI site, previously used to clone human Bcl-2 cDNA, to clone the PCR-amplified cDNA encoding zFP . . n( }, g( w5 @! e; l. s3 T5 R; K1 U3 S4 _" h* P0 u7 B, P- ]/ V0 o
Mice ? Transgenic mice were prepared by pronuclear microinjection of electroeluted DNA fragment containing H2K-zFP-i-LTR cassette into F1 of C57BLACK/6xC3H (Fig. 1B). Transgenic mice were genotyped by FACS screening of ACK-treated (hypotonic solution to remove erythrocytes) peripheral blood. Transgenic mice were back-crossed at least seven times onto BA mice (C57BL6/Ka-Thy1.1; Ly-5.1). Mice used in this study were 6–12 weeks old. All mice were maintained on acidified water (pH 2.5). : s* X! i" `& e) J& A6 B' \0 W0 r ) f& w' K8 ?& y6 i- B9 cIsolation of HSCs and Flow Cytometry Analysis ? All flow cytometry procedures were performed using the Vantage-SE FACS station at the Stanford Core FACS facility. For HSC enrichment, whole bone marrow was collected from the hind legs of H2K-zFP transgenic mice and enriched using c-kit–specific antibody as described . Briefly, after incubation with biotinylated c-kit–specific mAb (clone 3C11), cells were washed and incubated with streptavidin-conjugated magnetic beads (Miltenyi Biotec). Labeled cells were then enriched by passing the cells through a magnetic column (Miltenyi Biotec) and eluting the retained c-kit–positive cells after removing the column from the magnet. Magnetic enrichment for Sca-1–expressing bone marrow cells was performed similarly using PE-conjugated anti–Sca-1 mAb (clone E13-161-7) and anti-PE microbeads (Miltenyi Biotec). The brightest cells in the FITC channel were then double-sorted and used for injections. Dead cells were excluded by addition of propidium iodide (PI) or 7AAD and gating on the negative cells. The Hoechst 33342 (SP) analyses were performed exactly as described . Directly conjugated anti-mouse CD34-APC was purchased from BD Biosciences (San Diego, http://www.bdbiosciences.com).* U( j* J8 q* |' Z
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Reconstitution Analysis ? Multilineage reconstitution analyses were performed using peripheral blood as previously described . Briefly, syngeneic BA mice were lethally irradiated (970-Rad split dose) and retro-orbitally injected with the double-sorted zFPbright cells from H2K-zFP transgenic mice together with 3.5 x 105 syngeneic whole bone marrow cells. Peripheral blood from reconstituted mice was analyzed at 4, 8, and 32 weeks after reconstitution using B220 and CD3 markers for B and T cells, respectively (lymphoid), M1/70 GR1 markers for myeloid cells, and TER119 marker for early erythroid progenitors. Mice were maintained on antibiotics (1.1 g/l neomycin sulfate and 106 U/l polymyxin B sulfate) for at least 8 weeks after irradiation. Limiting dilution analysis was performed essentially as described . The zFPbright cells from H2K-zFP transgenic mice were prospectively isolated by double-sorting, with the second sort using the clone sort options of the Torbo Vantage FACS into the Terazaki plates. The presence of a given number of cells per well was confirmed by direct observation under the fluorescence microscope before mixing with 3 x 105 syngeneic whole bone marrow cells and retro-orbital injection. Twenty recipient mice were used for 1- and 2-cell reconstitution analysis, 10 mice for 5 cells, 15 mice for 10 cells, and 10 mice for 15 cells. Donor-derived cells were identify using the zFP expression (green cells). _" I" E" C- K; \; k 1 V1 E3 d# C: I: H c0 @ACKNOWLEDGMENTS ) P [$ q: |3 B! _2 R b! }, H5 S9 L
Available online without subscription with the open access option. ' d) c/ B3 H; _; Y! d& d$ K& S+ b3 I+ T! q6 C
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