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First published online August 9, 2005
Stem Cells Vol. 23 No. 10 November 2005, pp. 1617 -1625
doi:10.1634/stemcells.2004-0374; www.StemCells.com
© 2005 AlphaMed Press

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Simple and Efficient Isolation of Hematopoietic Stem Cells from H2K-zFP Transgenic Mice

Didier Surdeza, Beatrice Kunza, Amy J. Wagersc, Irving L. Weissmanc, Alexey V. Terskikha,b

a Department of Life Science, Swiss Federal Institute of Technology, Lausanne, Switzerland;
b The Burnham Institute, La Jolla, California, USA;
c Department of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, California, USA

Key Words. Hematopoietic stem cells • Fluorescent protein • Prospective isolation

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{at}Burnham.org


   ABSTRACT
Top
 Abstract
Introduction
 Results
 Discussion
 References
 
We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFPbright cells are also c-kithigh, Sca-1high, Linneg, Flk-2neg, which is a bona fide phenotype for long-term HSCs. Double-sorted zFPbright HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.


   INTRODUCTION
Top
 Abstract
 Introduction
Results
 Discussion
 References
 
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 [1], p27 [2], SCL [3], Bcl2 [4], Bmi1 [5], Bmpr1{alpha} [6], 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.

In the field of hematopoiesis, the prospective isolation of hematopoietic stem cells (HSCs) from adult mouse bone marrow [7, 8], followed by prospective isolation of lymphoid and myeloid progenitor populations [9, 10], allowed the gene expression analysis of these highly purified, functionally homogeneous cell populations [11, 12]. 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 [7, 8, 13]. More recently, long-term HSCs were isolated using a slightly different combination of markers, namely EndohighSca-1highLinneg/loworEndohighSca-1highRholow (Rhodamine-123 low) [14, 15]. 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.

More than 20 years ago, Visser et al. [16] 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.


   RESULTS
Top
 Abstract
 Introduction
 Results
Discussion
 References
 
Preparation of H2K-zFP Transgenic Mice
We used SMART cDNA subtraction (Clontech, Mountain View, CA, http://www.clontech.com/clontech) technology to identify transcripts enriched in short- versus long-term HSCs in C57BL6/Ka-Thy1.1 mice. The cell isolation and subtraction procedures were done as previously described [8, 17, 18]. The reverse transcription–polymerase chain reaction (RT-PCR) verification of the candidate transcripts confirmed the differential expression of several genes, including the Mac-1 marker, whose expression is a hallmark of short-term HSCs [8] (Fig. 1AGo). We have noticed that the H2K gene transcript is enriched in long-term as opposed to short-term HSCs (Fig. 1AGo). Using the promoter/enhancer element of H2Kb gene, which was previously used to efficiently express human BCL2 protein in HSCs [4], we generated a transgenic mouse line expressing a novel GFP, Zoanthus FP500 from Anthozoa species, or zFP (kindly provided by Dr. Sergey Lukyanov, Institute of Bioorganic Chemistry, Moscow). The excitation and emission properties of zFP protein are very close to those of GFP (488-nm excitation laser, fluorescein isothiocyanate [FITC] detection channel), but zFP is more stable and less toxic than GFP ([19] and A.V.T., unpublished observations). In addition to the H2Kb promoter, the transgenic construct contains two H2Kb introns and the Moloney MuLV enhancer/poly(A) element (Fig. 1BGo).



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  		Figure 1. (A): Semiquantitative reverse transcription–polymerase chain reaction analysis of long-term (L) and short-term (S) hematopoietic stem cells (HSCs). ß-Actin and ubiquitin were used as housekeeping controls; c-kit marker is known to be expressed at high levels in both long- and short-term HSCs, whereas Mac-1 is a hallmark of short-term HSCs. The number of polymerase chain reaction cycles is indicated below each pair of long- and short-term HSCs. (B): The H2K-zFP transgenic construct depicting the H2Kb-promoter/enhancer element H2Kb intron and Moloney MuLV enhancer/poly(A) site as well as the exon/intron structure of putative mRNA.

 
Characterization of the Promoter Activity
To assess the promoter activity, we have examined several tissues known to express the H2Kb protein. As expected, zFP expression was found in all tissues examined as judged by green fluorescence of tissue sections (Fig. 2Go). Lymphoid areas of the spleen known to express a higher level of H2K compared with myeloid areas could be clearly distinguished (Fig. 2Go, spleen stars). Various intensity patterns were observed in lungs, kidney, liver, and thymus, whereas muscles seemed to be more homogeneously positive (Fig. 2Go). In this study, however, we will focus only on the zFP expression in the hematopoietic system. We took advantage of the zFP fluorescence to correlate the expression from the 2-kB promoter element and the endogenous H2Kb promoter. The cells from blood, lymph nodes, spleen, bone marrow, and thymus were isolated and stained with the biotinylated anti-H2Kb antibody followed by avidin-allophycocyanin (APC) conjugate. With the exception of thymus, the flow cytometry analysis of blood, lymph nodes, spleen, and bone marrow showed that most zFP-positive cells also express H2Kb surface protein (Fig. 3Go).



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  		Figure 2. Activity of H2Kb promoter fragment (zFP fluorescence) in different tissues. Fresh-frozen sections (10 µM) were prepared from spleen, lungs, kidney, muscle, liver, and thymus and examined under the fluorescence microscope using the standard green fluorescent protein filter set. Asterisks indicate lymphoid areas that express high H2K levels.

 


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  		Figure 3. Comparison of the H2Kb promoter element-driven zFP expression with the level of endogenous H2Kb surface protein in blood, lymph nodes (LN), spleen, bone marrow, and thymus. The 5% probability contour plots are shown.

 
Whole Bone Marrow Analysis
We then analyzed whole bone marrow from H2K-zFP transgenic mice. No difference was found between wild-type and transgenic animals in the number of T cells (CD3, CD4, and CD8 markers), B cells (B200, CD19), myeloid cells (M1/70, GR1), and erythroid progenitors (TER119 marker) in the bone marrow, spleen, or lymph nodes analyzed (data not shown). Also, no difference was found in the number of HSCs, identified by staining for c-kit, Sca-1, Lin (lineage cocktail containing CD3, CD4, CD8, CD5, B220, M1/70, GR1, TER119) (Fig. 4AGo and data not shown). Most of the cells in all lineages analyzed (with the exception of TER119+ cells) were positive for zFP (Fig. 4CGo). Consistent with the levels of H2K protein expression on different hematopoietic cells, the lymphocytes (B220+, CD4+, CD8+) expressed higher levels of zFP compared with myeloid (Mac1/GR1+) and early erythroid (TER119+) compartments.



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  		Figure 4. Analysis of stem cell compartment in wild-type (w.t.) and H2K-zFP transgenic bone marrow. (A): Most c-kithigh Sca-1high cells from the transgene are Linneg and zFPhigh. (B): Increased frequency of c-kithigh Sca-1high cells in zFPbright cells from the whole bone marrow. The 5% probability contour plots with outliers are shown. (C): Most lineage-positive cells (with the exception of early erythroid TER119+ cells) are positive for zFP marker. The zFP intensity histograms of the propidium iodide–excluded, corresponding marker-positive cells are shown. (D): The staining profile of c-kit and Sca-1 versus zFP; 5% probability contour plots are shown. Abbreviations: APC, allophycocyanin; PE, phycoerythrin; TxRd, Texas Red.

 
When the c-kithigh, Sca-1high cells in transgenic bone marrow were analyzed for zFP expression, a major Linneg, zFPhigh population was observed (Fig. 4AGo). Thus, more than 60% of the c-kithigh, Sca-1high, zFPhigh cells in transgenic bone marrow are also Lin negative, suggesting that HSCs from H2K-zFP transgenics will have high levels of zFP expression. In other words, most cells expressing the classic markers for HSCs (here both long- and short-term) are also highly positive for zFP. However, when zFPbright (the brightest zFP cells, inthiscase, 0.2%) were analyzed, only approximately 5% of these cells were found to be c-kithigh, Sca-1high. This result suggests that in addition to HSCs, other cells express high levels of zFP in the unfractionated whole bone marrow of H2K-zFP transgenics. On the other hand, this zFPbright cell population from transgenic total bone marrow contains many more HSCs (a conservative estimate of ~1%–2% because ~5% of zFP high are c-kithigh, Sca-1high and more than 60% of these cells are Lin negative [Figure 4Go]) than the overall frequency (~0.01%)of HSCs in unfractionated wild-type C57BL6 or transgenic marrow [8]. Thus, a simple selection for zFPbright cells allows a 100- to 200-fold enrichment for HSCs compared with unfractionated bone marrow.

Analysis of the c-kit–Enriched Bone Marrow
In practice, the isolation of large numbers of HSCs that is usually required for gene transfer experiments always necessitates an enrichment step to optimize the FACS time. We have used the c-kit enrichment procedure that is well-established in our laboratory (see Experimental Protocols for details) based on the fact that all HSCs are included in the c-kithigh fraction of the bone marrow whereas most cells expressing various lineage markers are c-kitneg/low [8, 20]. One can thus use two non-cross-reacting mAbs specific for the c-kit marker, one biotinylated for enrichment and another labeled with a fluorescent dye. The c-kit enrichment of bone marrow usually results in increasing the frequency of the HSC population to 0.1%–1% of enriched bone marrow.

We analyzed c-kit–enriched bone marrow from H2K-zFP transgenic mice. Similar to the situation in whole bone marrow, most c-kithigh, Sca-1high cells were found to be zFPbright and Linneg/low (Fig. 5AGo). Yet in contrast to that seen in total bone marrow, most zFPbright cells (~0.2% of the brightest cells) are also c-kithigh, Sca-1high, suggesting that the zFPbright cells in the enriched bone marrow have the phenotype of HSCs (Fig. 5BGo). The threshold limit of approximately 0.2% zFPbright cells was found empirically; increasing this number will result in progressive dilution of the HSC phenotype (c-kithigh, Sca-1high cells) and the acquisition of lineage-positive cells. However, if one considers only the Linneg cells, the levels of zFP expression are likely to correlate with different progenitor populations (see below).



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  		Figure 5. Analysis of c-kit–enriched bone marrow from H2K-zFP transgenic mice. (A): Most c-kithigh Sca-1high cells from enriched transgenic bone marrow are Linneg. Note that most zFPbright cells are Linneg. (B): The brightest zFP cells from enriched transgenic bone marrow are highly enriched for Linneg, c-kithigh, Sca-1high cells. (C): The brightest zFP cells from enriched transgenic bone marrow are Flk-2neg and highly enriched for c-kithigh, Sca-1high. The 5% probability contour plots with outliers are shown. Abbreviations: APC, allophycocyanin; PE, phycoerythrin; TxRd, Texas Red.

 
We took advantage of the recent observation that long-term HSCs are Flk-2–negative within the c-kithigh, Sca-1high, Linneg population[13]. In a separate experiment, we analyzed the zFPbright cells from c-kit–enriched transgenic bone marrow and found that they are Flk-2–negative (Fig. 5CGo, left panel). Furthermore, analysis of Flk-2neg/zFPbright cells revealed that most are c-kithigh and Sca-1high (Fig. 5CGo, right panel). Indeed, this is the same zFPbright population as seen in Figure 4BGo, right panel. Thus, we have demonstrated that more than 60% of the zFPbright cells from the enriched bone marrow of H2K-zFP transgenics are similar to the long-term HSCs in that the yarec-kithigh, Sca-1high, Flk-2neg[13]. The exact proportion of cells with the HSC phenotype within the zFPbright cell population varied in our hands (the best case, 94%, presented in Fig. 5BGo, right panel; the less favorable case, 60%, presented in Fig. 5CGo, right panel). These variations are most likely due to the known fluctuations in the efficiency of c-kit enrichment on the magnetic columns and variations in antibody staining.

To further scrutinize the phenotype of zFPbright cells, we analyzed their relationship with side population (SP) as originally described by Goodell and colleagues [21, 22]. We compared the mean zFP fluorescence in total c-kit–enriched bone marrow, the entire SP population, and separately in the tail and top fractions of SP (Figs. 6A–6CGo). On average, approximately 37% of all zFPbright cells were found in the SP fraction, and 16% of cells in the SP fraction were also zFPbright (Figs. 6A, 6BGo). The mean zFP fluorescence in SP was approximately 10-fold higher than average (2456 vs. 264). The SP tail fraction is thought to contain primitive HSCs with a surprisingly high homing capacity [23]. We found that the SP tail fraction, which comprises 0.07% of all cells (compared with 0.47% of cells in total SP fraction of c-kit–enriched bone marrow) contained the brightest zFP cells, having a mean fluorescence of 4063 in SP tail versus 2212 in SP top fraction (Fig. 6CGo). Furthermore, the proportion of SP tail cells increased with zFP brightness (Fig. 6DGo). For instance, almost half (43%) of cells were found in SP tail fraction when analyzing the top 0.02% of the brightest zFP cells (Fig. 6DGo). As previous observations indicate that true primitive HSCs are negative for the CD34 marker [23, 24], we analyzed the expression of CD34 antigen in zFPbright cells in c-kit–enriched bone marrow. Staining with CD34 antibody revealed that 96% of zFPbright cells are CD34-negative(Figs. 6E, 6FGo). Collectively, these data are consistent with idea that the zFPbright population is highly enriched in HSCs, with the brightest zFP cells consistently found in the SP tail fraction, which has been shown to be enriched for the most primitive HSCs having exceptionally high homing activity [23].



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  		Figure 6. Relationship of zFPbright cells to other hematopoietic stem cell markers in c-kit–enriched transgenic bone marrow. (A): Representative dot plot of Hoechst 33342 staining of c-kit–enriched H2K-zFP bone marrow; histogram analysis of zFP fluorescence in total SP fraction (top + tail). The zFPbright cells (0.2%) gated out by the red line represent 16% of total cells in SP fraction. (B): Distribution of zFPbright cells (0.2%) within SP fraction; percentages of zFPbright in SP-top and SP-tail are indicated. (C): Mean zFP fluorescence in c-kit–enriched H2K-zFP bone marrow, total SP fraction, SP-tail, and SP-top fractions. (D): Distribution of various-brightness zFP cells (gated from top 0.02% to top 1%) within the SP fraction; the top 0.2% gated population (called zFPbright cells in the current study) is denoted by the red square. (E, F): Dot plot and histogram analysis of CD34 expression revealed that ~96% of zFPbright cells are negative for CD34 marker. Abbreviations: APC, allophycocyanin; BM, bone marrow; SP, side population.

 
In Vivo Reconstitution with zFPbright Cells
Next, we examined the in vivo reconstitution potential of zFPbright cells from the bone marrow of our transgenic mice. The 0.2% of zFPbright cells from the c-kit–enriched bone marrow were isolated by flow cytometry and injected along with 3.5 x 105 syngeneic bone marrow cells into lethally irradiated recipients. In the pilot experiment, we observed long-term reconstitution (10 weeks) in all three groups (450, 150, and 50 cells per animal; three animals per group) of injected mice (data not shown). We then performed a second experiment, decreasing the number of injected of cells to 80, 40, and 20 cells per animal. Eight weeks after the reconstitution, all animals injected with 80 and 40 cells and four out of five animals injected with 20 cells were reconstituted (Fig. 8AGo). HSCs also express Sca-1 antigen at high levels [7, 8]. In parallel, we used an alternative method for HSC enrichment from bone marrow, namely using the Sca-1 antigen. In this case, we enrich for Sca-1–positive cells using phycoerythrin (PE)–conjugated Sca-1 antibody and anti-PE-conjugated magnetic beads. The brightest 0.2% of cells were isolated by FACS and injected into lethally irradiated recipients at 100 cells per mouse. Ten weeks later, a robust multilineage reconstitution was observed in all animals (Fig. 8BGo). These results suggest that both procedures, aimed at the elimination of lineage-positive cells, achieve a cell population in which the zFPbright cells are highly enriched in long-term HSCs.



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  		Figure 7. Correlation between H2Kb (zFP) expression within the Linneg cells and the Sca-1 marker expression in H2K-zFP transgenic bone marrow. The percentage of zFP/Linneg cells within the gated area is indicated. Note the unchanged high level of the c-kit marker in all cases, suggestive of various progenitor populations. The 5% probability contour plots with outliers are shown.

 


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  		Figure 8. In vivo reconstitution analysis of the double-sorted zFPbright population from H2K-zFP transgenic mice. (A): The indicated numbers of cells from c-kit–enriched transgenic bone marrow were assayed. Peripheral blood chimerisms at 8 weeks are shown. The zFP-positive cells were scored using the following markers: lymphoid B200 + CD3, myeloid M1/70 + GR1, erythroid TER119. (B): zFPbright cells were isolated from Sca-1–enriched BM and used for reconstitution analysis, similarly to that in (A). (C): Classic limiting dilution analysis; the dashed line indicates the number of cells resulting in 37% engraftment failure. Animals with 1% chimerism (zFP+ cells) and above were scored as positive; the background detection level of 0.05% of zFP+ cells is indicated by dotted horizontal bar in (A) and (B). Abbreviation: BM, bone marrow.

 
Finally, we performed a classic limiting dilution analysis by reconstituting mice with a precisely controlled number of double-sorted zFPbright cells. The presence of a given number of cells in a Terasaki well was confirmed for each well by direct observation using an inverted fluorescent microscope (Olympus IX81; Olympus Schweiz AG, Volketswil, Switzerland, www.olympus.ch) before injections. The Poisson statistics suggest that positive-engrafting mice receiving low cell doses, of whom more than 63% are in fact negative for engraftment, are positive due to the expansion of one cell clone and, thus, the 37% of engraftment failure defines the limit dilution dose [25–29]. The estimated limit dilution dose for zFPbright cells was approximately 12 cells, which is comparable to the approximately 10- to 15-cell limit dilution dose previously reported for HSCs isolated using conventional multicolor flow cytometry [8, 15, 24].

zFP Expression Correlates with Sca-1 Expression
While analyzing the data, we made a serendipitous observation that correlated z FP and Sca-1 expression levels (Fig. 7Go). It was previously demonstrated that both lymphoid- and myeloid-committed progenitors are Linneg and express high levels of c-kit but low levels of Sca-1 [9, 10, 30]. Similarly, we noted that the level of zFP expression within the Linneg population is correlated with the level of Sca-1 expression whereas the c-kit expression level remains high (Fig. 7Go). It is tempting to speculate that the zFPlow, Linneg population, which is c-kithigh and Sca-1low, includes various types of committed progenitors but excludes HSCs and terminally differentiated cells. This hypothesis is currently under investigation.


   DISCUSSION
Top
 Abstract
 Introduction
 Results
 Discussion
References
 
The 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.

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 [8, 13]. 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.

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 [8]. 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.

A method to enrich for pluripotent HSCs described by Visser et al. [16] 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 [16]. 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. 3Go, bone marrow).

The cells in SP population, originally described by Goodell et al. [21], 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. [23] 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 [23, 24]. 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.

Previously, several groups used an enhanced GFP (EGFP) marker under the control of the Sca-1 promoter to enrich for HSC/ progenitor populations [31, 32]. However, a relatively modest enrichment of approximately 100-fold (meaning approximately 1 HSC in 100 cells) was achieved in one case [31] and a minimum of only 750 GFP+ cells were tested in the other case [32]. 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 [31, 32].

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.

Although 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 [19, 33], especially the red fluorescent proteins DsRed, fluorescent timer, fast DsRed, monomeric DsRed, and the far-red fluorescent protein [19, 34–37] to visualize the cells transformed with the desired vector.

Experimental Protocols

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 [4]. We used the NotI site, previously used to clone human Bcl-2 cDNA, to clone the PCR-amplified cDNA encoding zFP [19].

Mice   Transgenic mice were prepared by pronuclear microinjection of electroeluted DNA fragment containing H2K-zFP-i-LTR cassette into F1 of C57BLACK/6xC3H (Fig. 1BGo). 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).

Isolation 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 [38]. 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 [21, 22]. Directly conjugated anti-mouse CD34-APC was purchased from BD Biosciences (San Diego, http://www.bdbiosciences.com).

Reconstitution Analysis   Multilineage reconstitution analyses were performed using peripheral blood as previously described [9]. 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 [9]. 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).


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Results
 Discussion
 References
 
We are pleased to thank Julie Christensen for the microinjection of transgenic constructs and Holger Karsunky and Paola Bonfanti for their help with the data collection and tissue processing. A.V.T. was a recipient of the Irvington Institute fellowship. A.J.W. was supported by a fellowship from the American Cancer Society (grant No. PF-00-017-01-LBC). This study was partially supported by an NIH grant to I.L.W.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   FOOTNOTES
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 Abstract
 Introduction
 Results
 Discussion
 References
 
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   REFERENCES
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 Abstract
 Introduction
 Results
 Discussion
 References
 

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Received on December 29, 2004; accepted for publication on May 27, 2005.




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