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Stem Cells 2002;20:241-248 www.StemCells.com
© 2002 AlphaMed Press

A New Assay Method for Late CFU-S Formation and Long-Term Reconstituting Activity Using a Small Number of Pluripotent Hemopoietic Stem Cells

Guoxiang Yanga,b, Hiroko Hishaa, Yunze Cuia, Tianxue Fana, Tienan Jina, Qing Lia, Zhexiong Liana, Naoki Hosakaa, Yulin Lib, Susumu Ikeharaa

a First Department of Pathology, Transplantation Center, and Regeneration Research Center for Intractable Diseases, Kansai Medical University, Moriguchi City, Osaka, Japan;
b Department of Pathology, Norman Bethune Medical University, Changchun, China

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

Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara{at}takii.kmu.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously reported that Lin-/CD71-/MHC class Ihigh/c-kit<low bone marrow cells (c-kit<low cells) are pluripotent hemopoietic stem cells (P-HSCs), since they have the capacity to self-renew for at least 2 years in mice and differentiate into all hemopoietic lineage cells over the long term when serial bone marrow transplantation is carried out using 500 c-kit<low cells. In addition, we have found that the c-kit<low cells do not form colony-forming units-spleen (CFU-S) on days 8 to 14 but form late CFU-S (after 16 days). In the present study, to confirm that c-kit<low cells are truly P-HSCs, we examine whether a few (<=50) c-kit<low cells can form late CFU-S and reconstitute lethally irradiated recipients. We have established a new method to rescue lethally irradiated mice by transplantation of a few cells so that they survive for more than 16 days: 0.2 ml of 20 Gy-irradiated peripheral blood (PB) was injected into the recipients every 3 days. All the mice that had been transplanted with 25 or 50 c-kit<low cells alone died within 12 days, and no CFU-S were detected in their spleens. However, when 25 or 50 c-kit<low cells were injected and 0.2 ml of 20 Gy-irradiated PB was injected every 3 days, the recipients survived, and a small number of CFU-S were detected after 16 days. About 40% of the recipients injected with 50 c-kit<low cells and about 15% of those injected with 25 c-kit<low cells survived for more than 6 months. Moreover, donor-derived multilineage cells were detected in all the hematolymphoid organs of the recipient mice. This new assay method using a small number of cells would be of great advantage for clarifying which cells are truly P-HSCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pluripotent hemopoietic stem cells (P-HSCs) are characterized as cells with the capacity to eternally self-renew and differentiate into all hemopoietic lineage cells. P-HSCs have been successfully isolated and purified on the basis of cell surface antigen expression, vital dye staining, and physical characteristics (cell size and density) [1–3]. It has been shown that the expression of c-kit molecules (the receptors for stem cell factor) is an important marker for P-HSCs. In earlier studies, P-HSCs in the adult mouse liver [4] and bone marrow (BM) [5–8] were reported to be c-kit+. However, recent mouse and human studies have demonstrated that P-HSCs are c-kitlow; mouse hemopoietic progenitors (in the dormant stage) and human CD34+ primitive progenitors are enriched in c-kitlow cells when assayed by colony formation and long-term culture-initiating cells (LTC-ICs) [9,10]. Kawashima et al. have also shown that CD34+/c-kitlow human bone marrow cells (BMCs) can survive more than 18 months when engrafted in sheep BM [11].

We have previously reported that Lin-/CD71-/major histocompatibility complex (MHC) class Ihigh/c-kit<low BMCs (c-kit<low cells) are P-HSCs; we named them c-kit<low cells, since the phenotype is c-kit- but the c-kit message is only detectable by reverse-transcriptase polymerase chain reaction (RT-PCR). The c-kit<low cells have the capacity not only to generate multilineage cells but also to self-renew for more than 2 years after serial bone marrow transplantation (BMT), whereas c-kitlow cells could reconstitute only the first recipients [12]. The c-kit<low cells can also differentiate into all-lineage hemopoietic cells, even in the thymus when the cells are intrathymically injected [13]. In humans, c-kit+ cells are generated from c-kit<low cells of CD34+ cord blood cells after short-term culture in the presence of recombinant human interleukin-6 (IL-6), IL-7, FLT-3L, and immobilized anti-CD34 antibodies [14]; blastic cells with the c-kit<low phenotype become c-kitlow cells, then c-kithigh cells during a 5-day period in culture. c-kit<low cells (when more than 103 are injected into lethally irradiated recipients) form later (days 16-20) colony-forming units-spleen (CFU-S), although c-kit+ cells and c-kitlow cells form CFU-S on days 8-10 and days 12-14, respectively [15]. In addition, we have more recently found that mouse c-kit<low cells can differentiate into dendritic cells in culture [16]. These findings strongly suggest that c-kit<low cells are P-HSCs in both mice and humans.

In the present study, to confirm that c-kit<low cells are truly P-HSCs, we attempted to establish a new assay method for late colony formation and long-term reconstituting activity (LTRA) using a small number of cells. Using this method, we show that a small number (25 or 50) of c-kit<low cells can form late CFU-S (after 16 days) and reconstitute the recipients for more than 6 months when 0.2 ml of 20 Gy-irradiated peripheral blood (PB) is injected every 3 days until hemopoietic reconstitution.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female C57BL/6 (B6, Ly5.2) mice (6-8 weeks old) and male B6 Ly5.2 mice (10-12 weeks old) were purchased from CLEA Japan (Osaka, Japan; http://www.clea-japan.co.jp) and kept under specific pathogen-free conditions in our animal facility. Irradiated cells from male B6 Ly5.2 mice were used to rescue the lethally irradiated recipients. Congenic C57BL/6-Ly5.1-Pep3b (B6, Ly5.1) mice, which were obtained from The Jackson Laboratory (Bar Harbor, ME; http://www.jax.org), were bred and maintained in our animal facility.

Antibodies
Purified rat monoclonal antibodies (mAbs) against T cells (CD4, clone: GK1.5 and CD8, clone: 53-6.72), B cells (CD45R, B220, clone: RA3-6B2), granulocytes (Gr-1, clone: RB6), macrophages (CD11b, Mac-1, clone: M1/70), erythroid lineage cells (Ly76, clone: TER119), and transferrin receptor (CD71, clone: C2 [C2/F2]) were purchased from PharMingen (San Diego, CA; http://www.pharmingen.com). To deplete myeloid/lymphoid/erythroid lineage cells and cells in the cycling phase, these mAbs and anti-CD71 mAb were used in combination with magnetic beads conjugated with sheep anti-rat IgG Ab (Dynabeads® M-450; Dynal A.S.; Oslo, Norway; http://www.dynal.no). Fluorescein isothiocyanate (FITC)-coupled anti-H-2Kb mAb and phycoerythrin (PE)-coupled anti-c-kit mAb (clone: ACK4) from PharMingen were used to further purify the HSCs. FITC-coupled anti-Ly5.1 mAbs, PE-coupled mAbs against CD4, CD8, B220, Gr-1, Mac-1, and TER119, and biotin-coupled anti-Ly5.2 mAbs, which were also obtained from PharMingen, were used to analyze the surface phenotypes.

Purification of HSCs
Whole BMCs were collected from the femurs and tibias of B6 Ly5.1 mice that had been treated with 5-fluorouracil (5-FU, 150 mg/kg) 3 days before sacrifice. The BMCs were suspended in phosphate-buffered saline (PBS) containing 2% heat-inactivated fetal calf serum (FCS) and applied to Percoll (Pharmacia Fine Chemicals; Uppsala, Sweden; http://www.pnu.com) discontinuous density gradient centrifugation. After centrifugation, cells with a low density of 1.066 < p < 1.077 were collected, as reported previously [12,15]. The low-density cells were then incubated with a mixture of mAbs against CD4, CD8, Mac-1, Gr-1, B220, TER119, and CD71 for 30 minutes on ice, and then washed twice with PBS-FCS, followed by sheep anti-rat IgG-conjugated magnetic beads (Dynabeads®) to deplete the cells bearing myeloid/lymphoid/erythroid lineage markers and CD71 molecules. The Lin-/CD71- cells were then stained with PE-anti-c-kit mAb and FITC-anti-H-2Kb mAb, and MHC class Ihigh/c-kit- cells were sorted using a fluorescence-activated cell sorter (FACStar®, Becton Dickinson and Co. [BD]; http://www.bd.com). The sorted population showed low side scattering and moderate forward scattering and was about 0.008% of the 5-FU-treated whole BMCs (Fig. 1Go). We named the Lin-/CD71-/ class Ihigh/c-kit- cells as c-kit<low cells because, although phenotypically c-kit-, the c-kit message is only detectable by RT-PCR [12]. The sorted c-kit<low cells were used for transplantation. c-kitlow cells and c-kit+ cells were purified as previously described [6,10].



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  		Figure 1. FACS patterns of low-density/Lin-/CD71- cells stained with anti-c-kit and anti-H-2Kb Abs. A) The square gate shows the blast window (R1). B) c-kit and H-2Kb antigens are expressed on the blast window (R1). The cells in the R2 gate were sorted as c-kit<low cells.

 
Rescue of Recipients
In preliminary experiments, all mice that had received fractionated irradiation with (6.5 + 6.5) Gy (at a 4-hour interval) died within 14 days, and no CFU-S were detected in the spleens. In order to establish a method in which lethally irradiated mice could survive more than 16 days but in which no CFU-S were formed in the recipients' spleens, 10-20 Gy-irradiated PB, spleen cells, or BMCs were injected. The PB was collected from the heart of male B6 Ly5.2 mice, which had been sacrificed by cervical dislocation, using a 26-gauge syringe containing a small amount of heparin. The spleen cell suspensions were prepared by breaking up the spleens on a wire mesh, suspending with a pipette, setting out the clumps of debris, and washing. BMC suspensions were prepared by flushing the medullary cavity of the tibias and femurs using a 21-gauge syringe containing cold PBS-FCS. All i.v. administrations were performed using a 27-gauge needle to inject 0.2 ml of the PB or 0.3 ml (1 x 107 cells) of cell suspension via a tail vein or retroorbital sinus into mice anesthetized with metofane.

Transplantation of P-HSCs
Female B6 Ly5.2 recipient mice were lethally irradiated with (6.5 + 6.5) Gy 1 day before transplantation. The c-kit<low cells sorted from 5-FU-treated B6 Ly5.1 mice were i.v. injected into the lateral tail vein. The animals were maintained in a pathogen-free environment, and aqueous antibiotics were added to their drinking water for 3 days before irradiation and for more than 4 weeks after BMT. To support the survival of the recipients, rescue cells were supplied as described above. Mice transplanted with c-kit<low cells were monitored daily for survival.

CFU-S Assays
The recipient mice were killed on either day 16 or 20 after transplantation of the sorted c-kit<low cells, and their spleens were removed and fixed in Bouin's solution. Visible surface colonies were counted 1 day after fixation.

Analyses of Reconstitution
At various times after transplantation of the c-kit<low cells, the PB was collected from the tail vein of each mouse in heparinized tubes, and nucleated cells were separated using Ficoll gradients. The cells were incubated on ice for 30 minutes to be stained with a panel of PE-conjugated mAbs (anti-CD4, anti-CD8, anti-B220, anti-Gr-1, and anti-Mac-1), FITC-anti-Ly5.1 mAb, and Red 670-bio-anti-Ly5.2 mAb. The stained cells were analyzed using a FACScan® (BD).

Six months after transplantation, the recipient mice were sacrificed, and donor-derived Ly5.1+ cells with lineage-specific markers were identified in the spleen, thymus, lymph nodes, BM, and PB using a FACScan®.

Statistics
Intergroup variation in the survival rate (Fig. 2Go) and the percentage of donor-type cells (Fig. 3Go) were analyzed by a log-rank test and a Student's t-test, respectively.



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  		Figure 2. Survival rates of B6 Ly5.2 mice after BMT. B6 Ly5.2 mice were irradiated with (6.5 + 6.5) Gy. The next day, the recipients were transplanted with 25 or 50 c-kit<low cells sorted from 5-FU-treated B6 Ly5.1 mice either with or without 0.2 ml of 20 Gy-irradiated PB from B6 Ly5.2 mice every 3 days for 1.5 months. Statistical analyses were carried out using a log-rank test (15-20 mice/each group). *p <0.25; **p <0.01.

 


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  		Figure 3. Increases in percentages of donor-type cells in recipient PB. The 25 or 50 c-kit<low cells sorted from 5-FU-treated B6 Ly5.1 mice were transplanted into lethally irradiated B6 Ly5.2 recipients along with a supply of 0.2 ml of 20 Gy-irradiated PB from B6 Ly5.2 mice every 3 days for 1.5 months. Cells in the PB of the recipients were stained with donor-specific anti-Ly5.1 mAb-FITC and recipient-specific anti-Ly5.2 mAb-bio-Red 670 at various time points from 20 to 180 days. The mean percentages of donor-type cells ± standard deviation of three to six mice are shown. (Student's t-test; *p <0.2; **p <0.05; ***p <0.01).

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Injection of 20 Gy-Irradiated PB Can Rescue Lethally Irradiated Mice
In order to rescue lethally irradiated mice and keep them alive for more than 16 days, we compared the ability of the irradiated PB, spleen cells, and BMCs to rescue the irradiated mice. In our preliminary experiments, mice irradiated with (6.5 + 6.5) Gy died within 13 days, and no CFU-S were found in their spleens. When 0.2 ml of 10 Gy-irradiated PB was injected every 3 days, 70% of the recipients survived for 16 days after irradiation, but CFU-S were found in all surviving mice (data not shown). However, all the recipient mice died within 15 days when 10 Gy-irradiated spleen cells or BMCs (5 x 106 or 107 cells/mouse) were injected every 3 days (data not shown). Therefore, we next compared survival rates using 20 Gy-irradiated PB, spleen cells, and BMCs. As shown in Figure 4Go, about 60% of the recipients, when injected with 0.2 ml of 20 Gy-irradiated PB every 3 days, survived to 20 days, and no CFU-S were detected in the spleens of these mice. The survival rate was similar to that of mice injected with 10 Gy-irradiated PB (data not shown). All the mice that had been injected with 107, 20 Gy-irradiated spleen cells or BMCs died within 15 days, as did the mice that had been injected with 10 Gy-irradiated spleen cells or BMCs (data not shown). These findings indicate that the irradiation dose of 20 Gy can completely eliminate all the CFU-S-forming cells, and that the PB is more effective than either spleen cells or BMCs. Therefore, in the following experiments, we injected 20 Gy-irradiated PB every 3 days to investigate whether a few c-kit<low cells could form late (days 16-20) CFU-S and have LTRA.



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  		Figure 4. Survival rates of B6 Ly5.2 mice supplied with irradiated cells obtained from various hematopoietic organs. B6 Ly5.2 mice were lethally irradiated with (6.5 + 6.5) Gy. From the next day, the mice were i.v. injected with 0.2 ml of 20 Gy-irradiated (1) PB, (2) 1 x 107 spleen cells, or (3) 1 x 107 BMCs from B6 Ly 5.1 mice every 3 days. The survival rates were evaluated up to day 20. Each group consisted of 15 to 17 mice.

 
A Few c-kit<low Cells Can Form Late CFU-S on Days 16 to 20
c-kit<low cells were purified from 5-FU-treated B6 Ly5.1 mice as described. The sorted c-kit<low cells (Fig. 1Go) were transplanted into (6.5 + 6.5) Gy-irradiated B6 Ly5.2 mice, and 0.2 ml of 20 Gy-irradiated PB was then injected into the recipients. CFU-S appeared for the first time on day 16, their formations became bigger and clearer on day 20. A small number of CFU-S (mean ± standard deviation = 2.89 ± 1.54) was detected in the recipients on day 20 after transplantation of 50 c-kit<low cells (Table 1Go); the counts were compatible with the CFU-S counts (22.25 ± 10.55) when transplanted with 103 c-kit<low cells [15]. In contrast, no CFU-S were found on days 16 to 20 in the mice injected with 20 Gy-irradiated PB alone every 3 days (Table 1Go). FACS analyses revealed that CFU-S colonies expressed the donor-type Ly5.1 antigen, and that the BM and PB also contained donor-type Ly5.1+ cells (data not shown). We confirmed that c-kitlow cells formed CFU-S on days 12 to 14, whereas c-kit+ cells formed CFU-S on day 10, as we had previously described [15]. Therefore, we used only c-kit<low cells for the subsequent experiments.


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  		Table 1. CFU-S (on day 20) after transplantation of c-kit<low cells
 
A Few c-kit<low Cells Have the Capacity to Reconstitute Recipients for the Long Term and to Generate Multilineage Cells
To determine whether a few c-kit<low cells have LTRA, 25 or 50 c-kit<low cells sorted from 5-FU-treated B6 Ly5.1 mice were transplanted into (6.5 + 6.5) Gy-irradiated B6 Ly5.2 mice with or without the supply of 0.2 ml of 20 Gy-irradiated PB (every 3 days for 1.5 months after transplantation). As shown in Figure 2Go, all the mice that had been transplanted with 25 or 50 c-kit<low cells without the supply of 0.2 ml of 20 Gy-irradiated PB died within 12 days. However, the mice that had been transplanted with 25 or 50 c-kit<low cells with the supply of 20 Gy-irradiated PB showed 15% and 40% survival rates, respectively. The recipients that had received 25 c-kit<low cells with the supply of 20 Gy-irradiated PB showed a similar recovery pattern to that of the recipients that had received 50 c-kit<low cells with 20 Gy-irradiated PB, although the percentages of donor-derived cells in the PB were significantly lower in the former than in the latter (Figs. 3 and 5GoGo). A small number of donor-derived myeloid cells and B cells were detected after 16 days, and a substantial number of multilineage cells (including T cells) were clearly detectable in the PB 2 months after transplantation (Fig. 5Go).



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  		Figure 5. Reconstitution of multilineage cells in PB of recipients. The 25 or 50 c-kit<low cells sorted from 5-FU-treated B6 Ly5.1 mice were transplanted into lethally irradiated B6 Ly5.2 recipients along with a supply of 0.2 ml of 20 Gy-irradiated PB from B6 Ly5.2 mice every 3 days for 1.5 months. Two and 6 months later, the cells in the PB of the recipients were stained with a panel of lineage-specific mAbs-PE and donor-specific anti-Ly5.1 mAb-FITC.

 
Six months after transplantation, cells from several hematolymphoid organs were double stained with a panel of mAbs against mature myeloid/lymphoid cells and anti-Ly5.1 mAb to identify donor-derived cells. Figure 6Go shows representative data for the recipients of 50 c-kit<low cells. Ly5.1+ donor-derived CD4+/CD8+ cells, B220+ cells, and Gr-1+/Mac-1+ myeloid-lineage cells were detected in the BM, spleen, thymus, and lymph nodes. Although some recipient-type cells still remained in the PB (Fig. 5Go), spleen, and lymph nodes, such cells were almost undetectable in the BM and thymus (Fig. 6Go). Thus, the donor-derived multilineage cells increased gradually and had replaced the recipient-type cells in all the hematolymphoid organs by 6 months after transplantation.



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  		Figure 6. Long-term multilineage cell reconstitution of recipients. The 50 c-kit<low cells sorted from 5-FU-treated B6 Ly5.1 mice were transplanted into lethally irradiated B6 Ly5.2 recipients along with a supply of 0.2 ml of 20 Gy-irradiated PB from B6 Ly5.2 mice every 3 days for 1.5 months. Six months later, cells from the various organs were stained with a panel of lineage-specific mAbs-PE and donor-specific anti-Ly5.1 mAb-FITC.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been reported that transplanting a limited number of purified HSCs can reconstitute lethally irradiated mice for a long time [17–23]. By supplying "compromised cells," it was previously reported that less than 50 P-HSCs had the capacity to completely rescue lethally irradiated recipients for at least 6 months posttransplantation [24]. These reports, however, did not precisely examine whether donor P-HSCs themselves can reconstitute hemopoiesis of the lethally irradiated recipients, since the compromised BMCs contain very few HSCs and progenitor cells, which not only provide radioprotection to rescue the recipients but also generate into multilineage cells to sustain the recipient's life over a longer term.

In the present study, we established a new method to rescue lethally irradiated mice by supplying 0.2 ml of 20 Gy-irradiated PB every 3 days. This method allows the hemopoietic potentiality of P-HSCs to be measured more directly and precisely by eliminating the influence of "compromised cells." There are several advantages to this method. First, 20-Gy irradiation of PB completely eliminates all the CFU-S-forming cells but preserves the ability to rescue lethally irradiated mice for more than 20 days when supplied every 3 days (Table 1Go and Fig. 4Go). Therefore, late CFU-S can be detected using a few P-HSCs by this method. It has also been reported that irradiation limits the proliferation of lymphocytes but preserves their ability to facilitate BMT [25,26]. Gratwohl et al. have shown that, after 20-Gy irradiation, BMCs and peripheral buffy coat cells still have the capacity to release growth factors [25]. Recently, Waller et al. have shown that donor spleen cells irradiated with more than 7.5 Gy can facilitate allogeneic BMT without causing graft-versus-host disease, although the capacity of the spleen cells to proliferate is disturbed [26]. The irradiated cells thus seem to have time-limited biologic activity in vivo. Second, 20 Gy-irradiated PB has no hemopoietic reconstitution activity. In our preliminary studies, when 20 Gy-irradiated PB was injected alone, there were fewer than 2% PB-derived cells in the recipient PB, spleen, and BM on day 16 after the first injection of PB. When the blood supply was stopped, PB-derived cells disappeared from all hemopoietic tissues within 1 week, and the recipients died within 2 weeks (data not shown). Third, in this study, we have found that 0.2 ml of the PB contains ~2 x 106 nucleated cells, but that this quantity of PB is more effective than 107 spleen cells or BMCs. Therefore, it is conceivable that serum proteins and nutrients in the PB, as well as the mature blood cells, are important for the short-term survival of the lethally irradiated recipients.

In this study, by supplying 20 Gy-irradiated PB to rescue lethally irradiated mice, we have found that about 40% of the recipients injected with 50 c-kit<low cells could survive for more than 6 months. Even when recipients were injected with 25 c-kit<low cells, 15% survived for that long (Fig. 2Go). Furthermore, donor-derived myeloid cells, T cells, and B cells were detected in the PB, BM, spleen, thymus, and lymph nodes of the recipient mice (Figs. 5 and 6GoGo). Moreover, donor-type c-kit<low/H-2high cells were found in the BM of the reconstituted mice (data not shown). These findings show that the transplantation of even only a few c-kit<low cells provides the ability to generate multilineage cells for over 6 months. The transplantation of 50 c-kit<low cells resulted in a higher level of donor-derived Ly5.1 cells in the PB of recipients than did the transplantation of 25 c-kit<low cells (Fig. 3Go). The percentages of multilineage cells that had developed from 50 c-kit<low cells were always higher than those from 25 c-kit<low cells at the various time points of monitoring (Fig. 5Go). These observations indicate that the reconstitution by c-kit<low cells is dose dependent: the c-kit<low cells themselves (not the irradiated PB) are responsible for the reconstitution of recipients. Uchida et al. report a similar observation: a higher dose of HSCs leads to more rapid and sustained engraftment in both syngeneic and allogeneic BMT [27].

CFU-S assays have been used to estimate the frequency of HSCs and progenitor cells. There are several reports to show that P-HSCs are enriched in day 12 CFU-S [7,28,29]. There are some reports indicating that purified P-HSCs form day 13 CFU-S when a limited number (<=100 cells) are transplanted [21,30,31]. It is well known that more primitive P-HSCs proliferate slowly, and that they do not form day 12 CFU-S [32,33]. We have recently found that 103 c-kit<low P-HSCs have the capacity to form day 16 to 20 CFU-S, although c-kit+/Sca-1+ HSCs form day 10 CFU-S [15]. In the present study, even when the number of c-kit<low cells was reduced to 50 cells, a few CFU-S (approximately three) were detected on days 16 to 20 (Table 1Go). Considering that the seeding efficiency (f factor) [34] and extinction coefficiency ({omega}) [35] in mice are 0.17 and 0.6, respectively, 50 P-HSCs should form approximately 5 CFU-S. Therefore, the c-kit<low cells still consist of a heterogeneous population. Our findings were very recently confirmed by Ortiz et al. [36]. Using countercurrent centrifugal elutriation, they have shown that more primitive P-HSCs are c-kit- cells that lack the expression of c-kit mRNA and its cell surface protein. It is assumed that the c-kit- P-HSCs differentiate into c-kit<low cells, which express c-kit mRNA but lack cell surface c-kit expression. The c-kit<low cells then differentiate into c-kitlow cells and then c-kit+ cells that express both c-kit mRNA and cell surface protein, as we have previously shown in human cord blood cells [14]. Finally, c-kit+ cells further differentiate into various committed progenitor cell populations. c-kit- cells do not form CFU-S on day 12 but form secondary CFU-S (pre-CFU-S) on day 12 after serial transplantation. However, Ortiz et al. have not indicated whether late CFU-S can be formed in the first recipient, although we have shown that c-kit<low P-HSCs can form CFU-S on day 16 [15]. The differences between Ortiz et al.'s data and ours may be due to the different cell populations, since their cell population contains a moderate number of CD4+ or Thy-1+ cells, whereas our c-kit<low P-HSC population does not contain any CD4+ or Thy-1+ cells. More recently, Wiesmann et al. showed that a rare population (~10%) of CD27- cells among the Rholow Sca-1+ c-kithigh Lin- population has higher in vivo stem cell activity than CD27+ cells [37]. In our c-kit<low population, we found that about 70% of the cells are CD27- (data not shown), which is very high in comparison with the findings of Wiesmann et al. (only 10% of CD27- cells in the Rholow Sca-1+ c-kithigh Lin- HSCs). Surface marker analyses of the c-kit<low cells show that most of them are CD34-, Thy1.2-, CD38+, or Sca-1+. We are now determining the best conditions for Hoechst 33342 staining of P-HSCs, although preliminary results have indicated that the proportion contained in the "side population" is larger in the c-kit<low cells than in the c-kithigh cells.

Our previous study [14] showed that human CD34+/ c-kit<low cells can differentiate into c-kitlow and then c-kit+ cells. Therefore, the c-kit<low cells are considered to be precursors of c-kithigh cells: when a c-kit<low cell (dormant cell) enters the cell cycle, the cell expresses c-kitlow and c-kit+, followed by proliferation. One cell may return to the dormant state (c-kit<low cell), while the other differentiates into various hemopoietic cells. If this is the case, c-kit<low cells are the most primitive cells, although half the c-kit+ cells can return to be dormant HSCs (c-kit<low cells).

The c-kit<low cells contain cobblestone area-forming cells for a short time, but not LTC-ICs (data not shown), probably indicating that most such primitive HSCs cannot proliferate in vitro and, therefore, a three-dimensional microenvironment is essential for their proliferation and differentiation.

In the present study, we have established a new method to rescue lethally irradiated recipients using a small number of cells. This method would greatly contribute to the resolution of which cells are truly P-HSCs. We are in the process of examining which cells (c-kit- or c-kit<low, CD38- or CD38+, CD34- or CD34+, and CD27- or CD27+) are real P-HSCs using experiments based on the LTRA (more than 2 years). We have also shown that 50 c-kit<low cells with repeated injection of 20 Gy-irradiated PB can rescue irradiated recipients, with only a 40% survival rate. To increase the survival rate of recipients and to decrease the number of c-kit<low cells used for reconstitution, we are now using short-term repopulating cells of recipients in conjunction with c-kit<low cells of donors.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Ms. S. Miura and Mr. F. Ishida for conducting FACS sorting. We also thank Mr. Hilary Eastwick-Field and Ms. K. Ando for manuscript preparation.

This work was supported by a grant from the "Haiteku Research Center" of the Ministry of Education, grant-in-aid for scientific research (B) 11470062, grants-in-aid for scientific research on priority areas (A)10181225 and (A)11162221, Setsuro Fujii Memorial, the Osaka Foundation for Promotion of Fundamental Medical Research, a grant from "Millennium" of the Science and Technology Agency, and also a grant from Japan Immunoresearch Laboratories Co., Ltd (JIMRO).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on November 12, 2001; accepted for publication on February 1, 2002.





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