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Intrathymically Injected Hemopoietic Stem Cells Can Differentiate into All Lineage Cells in the Thymus: Differences Between c-kit⁺ Cells and c-kit^<low Cells

^a First Department of Pathology, Kansai Medical University, Moriguchi City, Osaka, Japan;
^b Department of Environment Pathology, School of Preventive Medicine, Norman Bethune University of Medical Sciences, Changchun, China;
^c Department of Scientific Research, Changchun Research Institute of Biological Production, Ministry of Health, Changchun, China

Key Words. Pluripotent hemopoietic stem cells • Intrathymic injection • c-kit

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
To investigate whether hemopoietic stem cells (HSCs) can differentiate into all lineage cells even in the thymus, we injected two types of HSCs (c-kit⁺ and c-kit^<low cells) obtained from C57BL/6 Ly5.1 mice directly into the thymus of 7.5 Gy-irradiated C57BL/6 Ly5.2 mice. When c-kit^<low cells (low density/lineage^–/CD71^–/major histocompatibility complex class I^high/Sca-1⁺/Thy-1^low/ c-kit^<low) were injected, donor-derived (Ly5.1) cells were detected on day 8 after intrathymic (i.t.) injection, and the number reached a maximum on day 24 after injection. Granulocytes and macrophages were also detected on day 8 after injection. However, B220⁺ B cells were observed on day 13. Eighteen days after i.t. injection, the injected lobes showed red color due to the synchronous development of erythroid cells. Histological studies revealed the development not only of erythroid lineage cells but also of megakaryocytes in the thymus. In contrast, when c-kit⁺ cells were injected, a significant number of donor-derived cells were detected on day 5 after i.t. injection (three days earlier than in the case of c-kit^<low cell injection). The differentiation into erythroid lineage cells was also observed six days earlier than when c-kit^<low HSCs were injected. These findings suggest that c-kit^<low HSCs are more primitive than c-kit⁺ HSCs, although both can differentiate into all lineage cells after i.t. injection.

	Introduction

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All mammalian blood cells (including lymphocytes) originate from a common very small population of pluripotent hemopoietic stem cells (P-HSCs) located in the bone marrow (BM) [1, 2]. With the exception of T lymphocytes, which require a special thymic environment for maturation and selection, hemopoiesis takes place in the BM. Sustained T cell development in the thymus requires a continuous input of precursor cells from the BM [3]. In the past several years, a variety of experimental approaches have been adopted regarding analyses of the later stages of T cell development in the thymus [4, 5]. Intrathymic precursors were initially described as being CD3^–CD4^–CD8^– (triple negative) [6-8], and the earliest intrathymic progenitor cells were reported to express a low level of CD4 [9, 10]. The early progenitors, which exhibit the CD4^low CD3^–CD8^–CD44⁺CD25^– c-kit⁺ phenotypes and are termed CD4^low cells, have been shown to contain T, B, natural killer, and DC precursor activity, although they lack the ability to generate myeloid lineage cells [10-12]. Nevertheless, information on the phenotypes and developmental potential of circulating progenitor cells is limited. Particularly, it has remained an open question whether thymus-colonizing cells contain T lineage-restricted prethymic progenitors or exclusively represent multilineage precursors which are induced to develop along the T lineage pathway only after entry into the thymic microenvironment [13]. To address this question, a variety of experimental approaches have recently been adopted. Reconstitution of thymopoiesis in irradiated recipient mice from the BM has been analyzed in detail [14]. Since Goldschneidier et al. developed a direct injection method [15], precursor cells could be supplied directly to the thymus in a single pulse. Reconstitution of thymopoiesis in sublethally irradiated recipient mice from c-kit⁺ HSCs has been analyzed after intrathymic (i.t.) injection [11, 12, 16]. However, there is no report on the analysis of thymopoiesis after i.t. injection of c-kit^<low HSCs. We have very recently found that Lin^–/CD71^–/class I^high/c-kit^<low cells (but not c-kit^low cells) are pluripotent HSCs with long-term (>1.5 years) reconstituting activity [17-20]. These findings prompted us to inject the c-kit^<low cells (phenotypically c-kit^– but only detectable at the message level) directly into the thymus. In the present study, we show that c-kit^<low cells as well as c-kit⁺ cells can differentiate into multilineage cells (including erythroblasts and megakaryocytes) in the sublethally irradiated thymus after i.t. injection, although the differentiation from the c-kit^<low cells is delayed.

	Materials and Methods

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Mice
Congenic C57BL/6 Ly5.1-Pep3b (B6 Ly5.1) mice (8-12 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in our animal facility. Female C57BL/6 (B6 Ly5.2) mice (five weeks old) were obtained from Clea Japan, Inc. (Osaka, Japan) and used as recipients for i.t. transfer experiments.

Preparation of Cell Suspension
Mice were killed, and blood around the thymic tissues was carefully removed as described previously [21]. The thymuses were kept on cold phosphate-buffered saline (PBS) containing 2% fetal calf serum (FCS) and 0.01% w/v NaN3 (PBS-FCS-Az), and were dispersed in medium by pressing the fragments between two glass slides. Cell suspensions were passed through a cotton gauze to remove cell debris, and dead cells were then removed by Percoll density gradients. The nucleated cells in the suspension were counted, then washed twice with PBS-FCS-Az. The thymus cells were stained with various kinds of monoclonal antibodies (mAbs), as described below.

Antibodies
Rat mAbs against CD4 (GK1.5), CD8 (53-6.72), CD45R (B220, RA3-6B2), granulocytes (Gr-1, RB6), and macrophages (Mac-1, M1/70) were purchased from Pharmingen (San Diego, CA), and the mAb against erythroid lineage cells (TER119) was kindly donated by Dr. T. Kina (Chest Disease Institute, Kyoto University; Kyoto, Japan). These mAbs were used to deplete myeloid/lymphoid lineage 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 (Pharmingen) and phycoerythrin (PE)-coupled anti-CD71 mAb (Pharmingen) were used to further purify P-HSCs. FITC-rabbit anti-mouse µ chain Ab, biotinylated-anti-Ly5.2 mAb, and biotinylated-anti-Ly5.1 mAb were purchased from Coulter Corporation (Hialeah, FL), and streptavidin-Cy-Chrome was purchased from PharMingen. PE-coupled mAbs against Gr-1, Mac-1, B220, Thy1.2, TER119, CD117, CD25, CD44 and CD4, and Cy-Chrome-conjugated anti-CD8 mAb were also purchased from Pharmingen for analyzing cell surface phenotypes.

Isolation of Hemopoietic Stem Cells
c-kit^<low HSCs were purified from the adult BM of C57BL/6-Ly5.1-Pep 3b (B6 Ly-5.1) using a modification of a previously described procedure [17]. Briefly, BM cells (BMCs) were collected from mice three days after a single injection of 5-fluorouracil (150 mg/kg, i.v.; Kyowa; Hakko Kogyo Co. Ltd.; Tokyo, Japan). Low-density (LD) BMCs (1.060 p < 1.074 g/cm³) were isolated by discontinuous density gradient centrifugation using Percoll, and the cells were incubated with mAbs against lineage markers (Mac-1, Gr-1, B220, CD4, CD8 and TER119) for 30 min on ice and washed twice with PBS-FCS-Az, then incubated with sheep Ab-rat IgG-conjugated immunobeads (IBs) at 4°C for 30 min with gentle agitation at a 3:1 bead/cell ratio. IB-rosetted cells were removed using a magnetic particle concentrator. Nonrosetted cells were recovered and reincubated with the same number of beads mentioned above, achieving a 40:1 bead/target cell ratio, since most of the target cells had been removed. The remaining nonrosetted cells were considered as Lin^– cells. After the Lin- cells were incubated with FITC-anti-H-2K^b and PE-anti-CD71 for 30 min on ice, H-2K^{b high}CD71^– cells were sorted.

c-kit⁺ HSCs were purified using a modified method by Spangrude et al. [22]. Briefly, LD Ly5.1 BMCs were isolated by discontinuous density gradient centrifugation using Percoll, and the cells were then incubated with mAbs against lineage markers (Mac-1, Gr-1, B220, CD4, CD8, and TER-119) for 30 min on ice and washed twice with PBS-FCS-Az. They were then incubated with sheep Ab-rat IgG-conjugated IBs at 4°C for 30 min with gentle agitation at a 3:1 bead/target cell ratio. IB-rosetted cells were removed using a magnetic particle concentrator. Nonrosetted cells were recovered and reincubated with the same number of beads mentioned above, achieving a 40:1 beads/target cell ratio, since most of the target cells had been removed. After the Lin^– cells were incubated with FITC-anti-Sca-1 and PE-anti-c-kit Ab (anti-CD117Ab) for 30 min on ice, Sca-1⁺ c-kit⁺ cells were sorted.

Intrathymic Injection
Intrathymic injection was carried out according to the modified protocol described previously [15]. Briefly, recipient mice (C57BL/6 Ly5.2) were exposed to 7.5 Gy of gamma irradiation from a ¹³⁷Cs source (Gammacell 40 Exactor; Nordion International, Inc.; Kanata, Ontario, Canada) at a dose rate of 1.140 Gy/min four to six h before i.t. injection. Anesthetized mice were secured by cords while the sternum was cut, and various numbers of cells in 10 µl of PBS were injected into the right lobes of the thymuses; as controls, 10 µl of PBS or 10⁴ peripheral blood mononuclear cells (PBMNCs) in 10 µl of PBS were injected into the left lobes. The incisions were closed and the mice were allowed to recover in a warmed enclosure.

Thymic Reconstitution Assay
Thymuses were removed at various times after i.t. injection, and single cell suspensions were prepared for flow cytometric analyses. Cells were incubated with FITC-conjugated Ab against the recipient-type Ly5.2, together with either anti-Gr-1, anti-Mac-1, anti-B220, anti-Thy1.2, TER-119 or anti-CD117 mAbs, and incubated with biotinylated (with streptavidin-Cy-Chrome) Ab against the donor-type Ly5.1. The stained cells were then analyzed by flow cytometry, with gating out for Ly5.2 recipient cells; list mode file (1 to 5 x 10⁴) events were collected for determining the percentages of donor-derived cells.

All experiments were carried out five times or more. Reproducible results were obtained, and representative data are therefore shown in the figures.

	Results

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Purification of c-kit^<low HSCs
The method of purifying of P-HSCs is described in Materials and Methods. LD/Lin^– cells were gated with respect to the blast window. The thresholds were set to discriminate the CD71^–/H-2K^bhigh population ( Fig. 1A). The histograms ( Fig. 1B) show the expression of cell surface markers on LD/Lin^– BMCs (light shading) and isolated H-2K^bhigh/ CD71^– cells (dark shading). The sorted cells showed Sca-1⁺/ c-kit^<low/Ly5.1^intermediate ( Fig. 1B)/Thy1^low (data not shown) markers. We have recently shown that thus purified cells have the long-term (>1.5 years) reconstituting activity [19].

View larger version (32K): [in this window] [in a new window]   Figure 1. Preparation of c-kit<low pluripotent hemopoietic stem cells. A) LD/Lin– cells were gated with respect to the blast window, and CD71–/H-2Kbhigh cells were sorted. B) The histograms of the sorted cells show Sca 1+/c-kit<low/Ly5.1intermediate markers.

To confirm the findings of Spangrude and Scollay [22], 5 x 10³ c-kit⁺ HSCs from Ly5.1 mice were injected into the thymic lobes of Ly5.2 mice. As shown in Figure 2 , a significant number of donor-derived (Ly5.1) cells appeared on day 5 after i.t. injection of c-kit⁺ HSCs. The percentages increased and reached a maximum on day 20. We next injected 5 x 10³ c-kit^<low HSCs of Ly5.1 mice into the thymic lobes of Ly5.2 mice to compare the kinetics of c-kit⁺ HSCs. A significant number of donor-derived cells appeared on day 13 after the i.t. injection of c-kit^<low cells, and the percentages reached a maximum on day 24. This level was maintained until day 35, and from day 40 the percentage of donor-derived cells gradually decreased: 72.9% on day 40 and 41.7% on day 60.

View larger version (11K): [in this window] [in a new window]   Figure 2. Kinetics of donor-derived (Ly5.1) cell development in the thymus after i.t. injection of Ly5.1 c-kit<low or c-kit+ HSCs.

View larger version (14K): [in this window] [in a new window]   Figure 3. Kinetics of donor-derived myeloid and lymphoid cell development after i.t. injection of c-kit<low HSCs: T cells (—), B cells (—), myeloid cells (•—•); Ly5.1+ cells: whole donor-derived cells (—).

View larger version (25K): [in this window] [in a new window]   Figure 4. FACS profiles of donor-derived T cells, B cells, granulocytes, macrophages and erythrocytes after i.t. injection of Ly5.1 c-kit<low HSCs. The profiles of each cell are shown on the days when each cell count reached its maximum after i.t. injection.

View larger version (39K): [in this window] [in a new window]   Figure 5. Kinetics of donor-derived T cell development after c-kit<low HSC injection.

View larger version (90K): [in this window] [in a new window]   Figure 6. Macroscopic (A) and microscopic (B) observations in the thymus. A) A right thymic lobe which was injected with c-kit<low HSCs shows red due to the development of erythroid cells. B) Microscopic examinations using smear sections (top) and paraffin block sections (bottom) show erythroblasts (I), granulocytes (II), myeloblasts (III), macrophages (IV top) and a megakaryocyte (IV bottom).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have shown that LD/Lin^–/ CD71^–/class I^high/c-kit^<low cells purified from the (Ly5.1) mouse BM can reconstitute the thymus of sublethally irradiated Ly5.2 mice after i.t. injection of the cells; donor-derived Ly5.1 T cells, B cells, granulocytes, macrophages and megakaryocytes developed in the thymus. To our surprise, the thymus lobes injected with HSCs (but not PBMNCs) showed red color due to the development of erythroid cells on day 18 after i.t. injection ( Fig. 6A). These findings indicate that LD/Lin^–/CD71^–/class I^high/c-kit^<low cells are pluripotent HSCs, which can differentiate into all lineage cells even in the thymus.

It was previously reported that i.t. injection of c-kit⁺ cells could reconstitute the irradiated thymus [16]; donor-derived T cells were detected from day 15 after injection, and the number remained constant until day 65 after injection. However, in the present study, we have observed that donor-derived cells are detected from day 13 after injection and the number remains constant until day 35 after injection. These differences may be due to the following two factors: A) the different mouse strains used (Ly congenic mice in our research; Thy-1 congenic mice in other research efforts) and B) the different cells used (c-kit^<low cells in our research; c-kit⁺ cells in other research efforts). The authors did not mention the development of erythroid cells or megakaryocytes after i.t. injection of c-kit⁺ cells, although they injected a maximum of 3 x 10³ c-kit⁺ cells.

Spangrude et al. have reported that 7 x 10³ Thy-1^low/Lin^–/Ly5.2 cells can reconstitute the thymus of Ly5.1 mice [23]; they describe the development of megakaryocytes and erythroid-like cells. However, they do not mention if the cells were c-kit⁺, c-kit^low or c-kit^<low. In the present study using c-kit⁺ HSCs, we confirmed their findings: donor-derived cells appeared on day 5 and reached a maximum on day 17. These results were similar to our observations ( Fig. 2).

It should be noted that the differentiation of erythroid cells is synchronized, since the thymus lobes injected with c-kit^<low cells turned red only from day 18 to day 21 ( Fig. 6A). This synchronization indicates that LD/Lin^–/CD71^–/class I^high/c-kit^<low cells consist of a uniform population with the characteristics of P-HSCs, although we injected 5 x 10³ c-kit^<low cells. It takes approximately three weeks for P-HSCs to differentiate into erythroid cells in the thymus in vivo, although it is uncertain whether they can differentiate into erythroid cells in the BM faster than in the thymus. It should be noted that erythroid cell differentiation from c-kit⁺ HSCs is also earlier than that from c-kit^<low HSCs ( Fig. 2).

We have attempted to reconstitute the thymus by injecting small numbers of the c-kit^<low P-HSCs, and were able to do so by injecting 5 x 10² LD/Lin^–/CD71^–/class I^high/c-kit^<low cells but not less than 5 x 10² cells (data not shown). This may be due to the irradiation dose (7.5 Gy), which may be insufficient to eliminate host thymocytes including precursors, since it is conceivable that donor P-HSCs compete with the intrathymic precursors of the host to proliferate and occupy the thymus.

The reconstituted thymus showed the same percentage of T cell subsets (CD4⁺/CD8^–, CD4^–/CD8⁺, and CD4⁺/CD8⁺ cells) as the noninjected normal thymus ( Fig. 5), suggesting that c-kit^<low cells can normally differentiate into mature T cells, as seen in the normal thymus.

In conclusion, both c-kit^<low and c-kit⁺ P-HSCs can differentiate into all lineage cells even in the thymus if they are injected i.t. but not i.v. Since c-kit⁺ HSCs can differentiate into all lineage cells earlier than c-kit^<low HSCs, it seems that c-kit^<low HSCs are more primitive than c-kit⁺ HSCs.

	Acknowledgments

 
This work was supported in part by a grant for Experimental Models for Intractable Diseases from the Ministry of Health and Welfare of Japan, and grants-in-aid for scientific research (02152117), (02670162), (03454177), (08770409) from the Japan Ministry of Education, Science and Culture.

We thank Mr. Fujio Ishida (Research Center of Kansai Medical University, Osaka, Japan) for flow cytometry studies, Ms. Y. Tokuyama and Ms. M. Murakami for their technical assistance, and Ms. K. Ando for her help in the preparation of the manuscript.

	References

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