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c-kit^<low Pluripotent Hemopoietic Stem Cells Form CFU-S on Day 16

First Department of Pathology, Kansai Medical University, Moriguchi City, Osaka, Japan

Key Words. CFU-S • Pluripotent hemopoietic stem cell • c-kit • Mouse

Dr. Susumu Ikehara, First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan.

	Abstract

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Using Ly5 congenic mice, we characterized the early differentiation step of pluripotent hemopoietic stem cells. Lineage^– (Lin^–)/CD71^– cells in the bone marrow cells were separated into major histocompatibility complex (MHC) class I^high/c-kit^low and MHC class I^high/c-kit^<low populations from C57BL/6 Ly5.1 male mice. These two populations (1,000 cells) were transplanted into lethally irradiated (5.5 Gy x 2) C57BL/6 Ly5.2 female mice. Colony-forming unit in spleen (CFU-S) assays were carried out on days 10, 12, 14, 16, and 20. In the mice that received c-kit^low cells, CFU-S were first detected on day 12, and the CFU-S counts gradually increased. In contrast, no visible colony was detected until day 14 in the mice that received c-kit^<low cells; CFU-S were first observed on day 16. Donor-derived (Ly5.1⁺) cells, such as B cells, T cells, and myeloid cells, were detected by fluorescence-activated cell sorter analyses, and donor-derived erythroid cells were detected by polymerase chain reaction analyses using Y-chromosome-specific primers. Donor-derived cells in the recipients of c-kit^low cells were detected in the spleen, bone marrow, and peripheral blood on day 12 after transplantation, while they were detected on day 16 in the mice that received c-kit^<low cells. Therefore, c-kit^<low cells have the capacity not only to form CFU-S on day 16 but also to reconstitute the recipients with donor-derived hematolymphoid cells 16 days after transplantation.

	Introduction

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Pluripotent hemopoietic stem cells (P-HSCs) are defined as cells with the capacity to eternally self-renew and differentiate into all hemopoietic lineage cells. Purification of P-HSCs has been carried out on the basis of the retention of rhodamine 123 [1, 2] and/or the expression of Sca-1 [3-6], Thy-1 [3-6], CD4 [7], Mac-1 [7], and CD34 [8]. The expression of c-kit molecules (the receptor for stem cell factor) has also been a marker to purify P-HSCs. In earlier studies, P-HSCs in the mouse bone marrow [9-12] and in the adult liver [13] have been reported to be c-kit⁺. However, recent mouse and human studies have provided evidence that P-HSCs are c-kit^low or c-kit^<low; mouse hemopoietic progenitors (in the dormant stage) and human CD34⁺ primitive progenitors are enriched in c-kit^low cells when assayed by colony formation and long-term culture-initiating cells [14, 15]. We have recently shown that P-HSCs are c-kit^<low (phenotypically c-kit^–, but the c-kit message is only detectable by reverse-transcriptase-polymerase chain reaction [RT-PCR]) by assessing their long-term repopulating activity after serial bone marrow transplantation (BMT). P-HSCs purified as Lin-/CD71-/MHC (major histocompatibility complex) class I^high/c-kit^<low cells (but not c-kit^low or c-kit⁺ cells) show the capacity not only to differentiate into multilineage cells but also to self-renew for more than 1.5 years [16]. Furthermore, we have more recently found that c-kit⁺ cells are generated from c-kit^<low cells of purified CD34⁺ cord blood cells after short-term culture [17]; blast cells with the c-kit^<low phenotype become c-kit^low cells, then c-kit^high cells during a five-day period in culture. These findings strongly suggest that the expression of c-kit molecules depends on the differentiation stage of HSCs, and that the c-kit^<low cells are the most primitive HSCs.

Colony-forming unit in spleen (CFU-S) assays have been used to estimate the frequency of progenitor cells; progenitors that form CFU-S on days 12 to 14 have been postulated to be more primitive than those that form CFU-S on day 8 [18]. It has, however, been reported that neither of these CFU-S represent the frequency of P-HSCs [19-23]. In the present study, using c-kit^<low P-HSCs, we have carried out CFU-S assays and have found that c-kit^<low P-HSCs form CFU-S on day 16. The expression or transition of c-kit molecules will be discussed in relation to the early differentiation of P-HSCs.

	Materials and Methods

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Mice
C57BL/6 (B6 Ly5.2) mice were purchased from CLEA Japan (Osaka, Japan) and maintained in our animal facility. The female mice were used as recipients for CFU-S assays. Congenic C57BL/6-Ly 5.1-Pep3b (B6 Ly5.1) mice obtained from The Jackson Laboratory (Bar Harbor, MA) were bled and maintained in our animal facility. The male mice were used as donors for CFU-S assays. All mice were used at 8-12 weeks of age.

Antibodies
Purified rat monoclonal antibodies (mAbs) against CD4 (GK1.5), CD8 (53-6.72), CD45R (B220, RA3-6B2), granulocytes (Gr-1, RB6), macrophages (Mac-1, M1/70), erythroid lineage cells (TER119), and transferrin receptor (CD71) were purchased from Pharmingen (San Diego, CA). These mAbs were used to deplete myeloid-/lymphoid-/erythroid-lineage cells and CD71⁺ cells in combination with magnetic beads conjugated with sheep anti-rat IgG Ab (Dynabeads^® M-450, Dynal A.S.; Oslo, Norway). Fluorescein isothiocyanate (FITC)-coupled anti-H-2k^b mAb and phycoerythrin (PE)-coupled anti-c-kit mAb (ACK4) from Pharmingen were used to further purify the P-HSCs. FITC-anti-Ly5.1 mAbs were purchased from Coulter Corporation (Hialeah, FL). PE-coupled mAbs against B220 (CD45R), CD4, CD8, granulocytes (Gr-1, RB6), macrophages (Mac-1, M1/70), and erythroid lineage cells (TER119), which were also from Pharmingen, were used to analyze the cell surface phenotypes.

Purification of HSCs
B6 Ly5.1 mice were treated with 5-fluorouracil (5-FU, 150 mg/kg) and, three days later, the bone marrow cells (BMCs) were collected and applied to Percoll (Pharmacia Fine Chemicals; Uppsala, Sweden) discontinuous density gradient. After centrifugation, cells with a density of 1.066 < p < 1.077 were collected, as reported previously [16, 24]. The low-density cells were treated with a mixture of mAbs against CD4, CD8, Mac-1, Gr-1, TER119, and CD71, followed by anti-rat IgG-conjugated magnetic-beads (Dynabeads^®) to deplete the cells bearing myeloid/lymphoid lineage markers and CD71 molecules. The cells, thus prepared (Lin^–/CD71^– cells), were stained with PE-anti-c-kit mAb and FITC-anti-H-2k^b mAb, and those cells with MHC class I^high/c-kit^low and MHC class I^high/c-kit^– (phenotypically c-kit^–, but only detectable at RT-PCR, therefore designated as c-kit^<low) were sorted using a FACStar^® (Becton Dickinson; San Jose, CA), ( Fig. 1, panel B). Both populations showed low side scattering and moderate forward scattering ( Fig. 1, panel A). Lin^–/CD71^–/class I^high/c-kit^low cells or Lin^–/CD71^–/class I^high/c-kit^<low cells were 0.006% or 0.008% of the 5-FU-treated whole BMCs, respectively. The purity of sorted cells was >99%.

View larger version (19K): [in this window] [in a new window]   Figure 1. Staining profiles of Lin–/CD71–/MHC class Ihigh/c-kit<low cells obtained from 5-FU-treated Ly 5.1 mice. Lin–/CD71– cells were stained with anti-H-2kb and anti-c-kit mAbs. Cells with moderate FSC and low SSC were gated (panel A), and cells with MHC class Ihigh/c-kit<low (panel B, gate A) and MHC class Ihigh/c-kitlow (panel B, gate B) were sorted using a FACStar. Panel C represents histograms of c-kit<low (sorted as gate A) and c-kitlow (sorted as gate B) cells.

Transplantation of P-HSCs
Recipient (B6 Ly5.2) mice were lethally irradiated (fractionated radiation: 5.5 Gy x 2 = 11 Gy, 4-h interval), and 24 h later were injected i.v. with 1,000 cells from the sorted c-kit⁺, c-kit^low, or c-kit^<low fraction. In a preliminary experiment, we found that some, but not all, mice that had received a single shot of 8.5 to 9 Gy irradiation alone (without transplantation of P-HSCs) formed internal CFU-S derived from radioresistant progenitors. Therefore, we used fractionated radiation (5.5 Gy x 2 = 11 Gy). In this condition, all the irradiated mice died within 14 days without the transplantation of P-HSCs, and no CFU-S were detected during these days.

CFU-S Assays
CFU-S assays were carried out on days 8, 10, 12, 14, 16, and 20. The spleens were removed and fixed with Bouin's fixing fluid before the spleen colonies were counted.

Flow Cytometric Analyses
Donor-derived Ly5.1⁺ cells with lineage-specific markers in the spleen, bone marrow, and peripheral blood were determined using a FACScan^® after staining with a panel of PE-conjugated mAbs (anti-B220, anti-CD4, anti-CD8, anti-granulocyte, anti-macrophage, anti-erythroid lineage cells) in combination with FITC-anti-Ly5.1 mAb.

Detection of Donor-Derived Cells in Spleen Colonies by PCR
Genomic DNA was isolated from the cells in the spleen colonies, and PCR amplification was performed for 30 cycles in PCR buffer containing dNTP and Taq polymerase (Perkin Elmer-Cetus; Branchburg, NJ), as described previously [24]. The following oligonucleotide primers with the sequences 5'-GCATTTGCCTGTCAGAGAGAG-3' (sense strand) and 5'-ACTGCTGCTGCTTTCCAACTA-3' (anti-sense strand) were constructed by and purchased from BIOLOGICA Co. (Nagoya, Japan). This primer pair was created to amplify a 411-bp region contained within the murine Y chromosome.

	Results

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CFU-S after Transplantation of c-kit^<low, c-kit^low, and c-kit⁺ Cells
c-kit^low or c-kit^<low cells (1 x 10³) from male B6 Ly5.1 mice were transplanted into allotype-disparate female B6 Ly5.2 mice, and CFU-S counts were measured 8 to 20 days later. As shown in Table 1, the recipients of c-kit^low cells formed visible CFU-S on day 12. CFU-S counts (and also spleen weight) gradually increased and became uncountable as a result of fusion on days 16 and 20. In contrast, no CFU-S were detected in the recipients of c-kit^<low cells on days 12 and 14, but first appeared on day 16. The numbers had increased on day 20, although each colony was small, as shown in Figure 2. Thus, c-kit^<low cells can form CFU-S, although colony formation is delayed.

View larger version (116K): [in this window] [in a new window]   Figure 2. CFU-S assays. The female C57BL/6 Ly5.2 mice that received 103 c-kit<low, c-kitlow, or c-kit+ cells were killed on days 8 to 20; c-kit<low and c-kitlow cells were obtained from the BMCs of 5-FU-treated male Ly 5.1 mice, and c-kit+ cells were obtained from the BMCs of male Ly 5.1 mice without 5-FU treatment. The spleens were removed and fixed with Bouin's fixing fluid before the spleen colonies were counted.

Development of Donor-Derived Cells
The development of donor-derived cells in the spleen colonies was confirmed by PCR analyses, as shown in Figure 3. Male (donor)-specific PCR products with 411 bp were clearly observed in the colonies of all mice that had received male B6 Ly5.1 c-kit^<low cells. Thus, the CFU-S in the recipients of c-kit^<low cells actually contained donor-derived cells carrying the male Y chromosome; the CFU-S on day 16 were not derived from the HSCs of the recipients. It is well known that erythroid-lineage cells expressing the molecules recognized by mAb TER119 do not express the Ly5 antigen [26]. Therefore, on day 16, we collected some CFU-S colonies that apparently contained erythroid-lineage cells and used a PCR assay to detect male (donor)-specific PCR products. Male-specific PCR products with the same 411 bp were clearly observed (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 3. Detection of donor-derived cells in the spleen colonies by PCR. Genomic DNA was isolated from the cells in the spleen colonies, and PCR amplification was performed for 30 cycles. The primer pair used was created to amplify a 411-bp region contained within the murine Y chromosome. Genomic DNA from female and male mice served as negative and positive controls, respectively. It is noted that all the recipients of c-kit<low cells contained donor-derived male cells that were confirmed by PCR, as shown in this figure.

View larger version (73K): [in this window] [in a new window]   Figure 4. Detection of donor-derived cells in the recipients. One thousand c-kit<low cells or c-kitlow cells from 5-FU-treated B6 Ly5.1 mice were transplanted into irradiated B6 Ly5.2 recipients. Eight to 20 days later, cells in the PB, BM, and spleen were stained with a donor-specific anti-Ly5.1 mAb (A). Figure 4B shows the representative FACS profiles of cells from the PB, BM, and spleens of recipients on day 16 after receiving c-kit<low cells. Cells were stained with various mAbs and anti-Ly5.1 mAb.

	Discussion

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c-kit, the receptor for steel factor or stem cell factor (SCF), has been known to be a marker of HSCs. In earlier studies of the expression of c-kit on hemopoietic progenitors, HSCs have been reported to be c-kit⁺ cells [9-11]. Recently, it has been reported that dormant primitive hemopoietic progenitors in mice (after treatment with 5-FU) and CD34⁺ primitive hemopoietic progenitors in humans are enriched in the c-kit^low cell population when assayed by colony formation in vitro in the presence of SCF, interleukin 3 (IL-3), and IL-11 [15] or by assessing long-term culture-initiating cells under the influence of IL-3, IL-6, SCF, GM-CSF, and erythropoietin [14], respectively. Recently, we have reported that Lin^–/CD71^–/MHC class I^high/c-kit^<low cells (approximately 0.01% of 5-FU-treated whole bone marrow cells) completely fulfill the definition of pluripotent HSCs: the capacity to generate multilineage cells and eternally self-renew, as shown by serial BMT assays [16]. Furthermore, we have more recently found that hematolymphoid cells derived from original donor (Ly5.1⁺) cells were detected even in the fifth recipients after serial BMT (Inaba M et al. manuscript in preparation). A recent report by Jones et al. [23] supports our findings. They have found that: A) lymphohemopoietic stem cells (LHSCs) with the phenotype of Lin^–/AA4.1^–/aldehyde dehydrogenase (ALDH)^–, isolated by counterflow centrifugal elutriation, produce delayed but long-term engraftment, and have the capacity to generate all lineage cells when 10 of these cells are transplanted; B) these LHSCs are small in size and, interestingly, actually c-kit^– at the cell surface, even at the mRNA level (by RT-PCR), and C) these LHSCs are capable of in vivo self-renewal when assayed by serial BMT [19], as reported in our previous paper. Furthermore, we have found that purified CD34⁺/c-kit^<low cells (phenotypically c-kit^– but only detectable at the message level) from human cord blood express c-kit molecules during the culture (c-kit^<low c-kit^low c-kit⁺) [17].

Jones et al. have reported that neither CFU-S (day 12) nor CFU-S (day 8) represent P-HSCs in mice and that progenitors isolated by counterflow centrifugal elutriation are different in size from cells generating CFU-S (day 8) or CFU-S (day 12) [22, 23]. However, they have not described whether the P-HSCs (Lin^–/AA4.1^–/ALDH^–/c-kit^– cells) form CFU-S on day 16. In the present study, we have clearly shown that c-kit^<low P-HSCs do not have the capacity to form either CFU-S (day 8) or CFU-S (day 12) but can form CFU-S on day 16 after transplantation ( Table 1 and Fig. 2). This indicates that Lin^–/MHC class I^high/c-kit^<low P-HSCs can form delayed CFU-S. Donor-derived cells (myeloid cells and B cells) were detected on day 16 (but not on day 14) in the PB, bone marrow, and spleen of the mice that received c-kit^<low cells ( Figs. 4A and 4B); donor-derived T cells were also detected in the PB, lymph node, and spleen of the recipients four weeks after the transplantation of c-kit^<low cells (data not shown).

As shown in Table 1 and Figure 2, it is likely that c-kit⁺ cells (obtained from mice without 5-FU treatment) are a mixture of c-kit^low and c-kit^high cells, since c-kit⁺ cells form CFU-S on days 10 and 12; we could not obtain c-kit⁺ HSCs (Lin^–/Thy-1^low/Sca 1⁺ cells) after 5-FU treatment. Very recently, Randall et al. have reported that the c-kit^– population, which corresponds to our c-kit^<low population, is a mystery population [27], since it does not form CFU-S on days 12 to 14 or days 8 to 10. The difference seems to be derived from the presence or absence of 5-FU treatment. Randall et al. obtained the c-kit^– population without 5-FU treatment. However, in our experience, no c-kit^<low or c-kit^low HSCs could be obtained without 5-FU treatment.

In conclusion, c-kit^<low cells have the capacity not only to form CFU-S on day 16 (as shown in the present study) but also to have an LTRA (>2 years) [16].

	Acknowledgments

 
Z.L. and B.F. contributed equally to this work.

We thank Mr. F. Ishida (Research Center of Kansai Medical University) for flow cytometry studies, and Ms. K. Ando for preparing the manuscript.

This work was supported by grants from the Ministry of Health and Welfare of Japan, the Ministry of Education, Science and Culture, and the Japan Private School Promotion Foundation.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Wolf NS, Kone A, Priestley GV et al. In vivo and in vitro characterization of long-term repopulating primitive hematopoietic stem cells isolated by sequential Hoechst 33342-rhodamine 123 FACS selection. Exp Hematol 1993;21:614-622.[Medline]

2. Spangrude GJ, Brooks DM, Tumas DB. Long-term repopulation of irradiated mice with limited numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function. Blood 1995;85:1006-1016.[Abstract/Free Full Text]

3. Spangrude GJ, Brooks DM. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood 1993;82:3327-3332.[Abstract/Free Full Text]

4. Uchida N, Weissman IL. Searching for hematopoietic stem cells: evidence that Thy-1.1^loLin^–Sca-1⁺ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J Exp Med 1992;175:175-184.[Abstract/Free Full Text]

5. Spangrude GJ, Brooks DM. Phenotype analysis of mouse hematopoietic stem cells shows a Thy-1-negative subset. Blood 1992;80:1957-1964.[Abstract/Free Full Text]

6. Chung LL, Johnson GR. Long-term hemopoietic repopulation by Thy-1^lo, Lin^–, Ly6A/E⁺ cells. Exp Hematol 1992;20:1309-1315.[Medline]

7. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994;1:661-673.[Medline]

8. Krause DS, Ito T, Fackler MJ et al. Characterization of murine CD34, a marker for hematopoietic progenitors and stem cells. Blood 1994;84:691-701.[Abstract/Free Full Text]

9. Orlic D, Fischer R, Nishikawa S-I et al. Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor. Blood 1993;82:762-770.[Abstract/Free Full Text]

10. Ikuta K, Weissman IL. Evidence that hematopoietic stem cells express c-kit, but do not depend on steel factor for their generation. Proc Natl Acad Sci USA 1992;89:1502-1506.[Abstract/Free Full Text]

11. Ogawa M, Matsuzaki Y, Nishikawa S et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991;174:63-71.[Abstract/Free Full Text]

12. Li CL, Johnson GR. Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization. Blood 1995;85:1472-1479.[Abstract/Free Full Text]

13. Taniguchi H, Toyoshima T, Fukao K et al. Presence of hematopoietic stem cells in the adult liver. Nat Med 1996;2:198-203.[Medline]

14. Gunji Y, Nakamura M, Osawa H et al. Human primitive hematopoietic progenitor cells are more enriched in KIT^low cells than in KIT^high cells. Blood 1993;82:3283-3289.[Abstract/Free Full Text]

15. Katayama N, Shih J-P, Nishikawa S-I et al. Stage-specific expression of c-kit protein by murine hematopoietic progenitors. Blood 1993;82:2353-2360.[Abstract/Free Full Text]

16. Doi H, Inaba M, Yamamoto Y et al. Pluripotent hemopoietic stem cells are c-kit^<low. Proc Natl Acad Sci USA 1997;94:2513-2517.[Abstract/Free Full Text]

17. Sogo S, Inaba M, Ogata H et al. Induction of c-kit molecules on human CD34⁺/c-kit^<low cells: evidence for CD34⁺/c-kit^<low cells as primitive hematopoietic stem cells. STEM CELLS 1997;15:420-429.[Abstract/Free Full Text]

18. Magli MC, Iscove NN, Odarchenko N. Transient nature of early hematopoietic spleen colonies. Nature 1982;295:527-529.[Medline]

19. Jones RJ, Celano P, Sharkis SJ et al. Two phases of engraftment established by serial bone marrow transplantation. Blood 1989;73:397-401.[Abstract/Free Full Text]

20. Ploemacher RE, Brons RHC. Separation of CFU-S from primitive stem cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: evidence for pre-CFU-S cell. Exp Hematol 1989;17:263-266.[Medline]

21. Bertoncello I, Hodgson GS, Bradley TR. Multiparameter analysis of transplantable hemopoietic stem cells. II. Stem cells of long-term bone marrow-reconstituted recipients. Exp Hematol 1988;16:245-249.[Medline]

22. Jones RJ, Wagner JE, Celano P et al. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 1990;347:188-189.[Medline]

23. Jones RJ, Collector MI, Barber JP et al. Characterization of mouse lymphohematopoietic stem cells lacking spleen colony-forming activity. Blood 1996;88:487-491.[Abstract/Free Full Text]

24. Ogata H, Bradley WG, Inaba M et al. Long-term repopulation of hematolymphoid cells with only a few hemopoietic stem cells in mice. Proc Natl Acad Sci USA 1995;92:5945-5949.[Abstract/Free Full Text]

25. Spangrude GJ, Scollay R. Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes; kinetics and phenotype of progeny. J Immunol 1990;145:3661-3668.[Abstract]

26. Scheid MP, Dennis T. Further description of the Ly-5 system. Immunogenetics 1979;9:423-431.

27. Randall TD, Weissman IL. Characterization of a population of cells in the bone marrow that phenotypically mimics hematopoietic stem cells: resting stem cells or mystery population? STEM CELLS 1998;16:38-48.[Abstract/Free Full Text]

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