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Stem Cells, Vol. 18, No. 4, 252-260, July 2000
© 2000 AlphaMed Press

Evidence of Peripheral Blood-Derived, Plastic-Adherent CD34–/low Hematopoietic Stem Cell Clones with Mesenchymal Stem Cell Characteristics

Ralf Hussa, Claudia Langeb,c, Eva M. Weissingerb,d, Hans-Jochem Kolbb,e, Karin Thalmeiera

a Institute of Pathology, University of Munich, Germany;
b Institute of Clinical Hematology, GSF, Munich, Germany;
c CellTec GmbH, Hamburg, Germany;
d Department of Hematology, University Hospital, Hannover, Germany;
e Department of Medicine III, Klinikum Grosshadern, Munich, Germany

Key Words. Hematopoiesis • Mesenchymal stem cell • CD34 • Fibroblast-like canine cells

Ralf Huss, M.D., Institute of Pathology, University of Munich, Thalkirchner Str. 36, D-80337 Munich, Germany; Telephone: 49-89-5160-4011; Fax: 49-89-5160-4043; e-mail: Ralf.Huss{at}lrz.uni-muenchen.de Received April 27, 2000; accepted for publication May 1, 2000.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hematopoietic system of vertebrates can be completely reconstituted with hematopoietic stem cells derived from the bone marrow, fetal liver, or cord blood, or even from peripheral-blood-derived cells. A cellular marker to identify those cells is the proteoglycan CD34, although we have shown that the earliest identifiable hematopoietic stem cell is a CD34– fibroblast-like cell which can differentiate into CD34+ hematopoietic precursors. Peripheral blood mononuclear cells were isolated from the heparinized blood of a dog and incubated in tissue culture in the presence of interleukin 6. After 10-14 days, an adherent layer of fibroblast-like cells had developed and cells were immortalized using the SV-40 large T antigen. Cells were cloned and subcloned by measures of limiting dilution, and various fibroblast-like clones were established. These fibroblast-like cells either do not express the CD34 antigen or express CD34 on a low level, although transcribing CD34. The CD34–/low cells express osteocalcin as a mesenchymal cell marker. The fibroblast-like cells eventually differentiate spontaneously in vitro into CD34+ precursors and show colony formation. Prior to autologous stem cell transplantation, one clone of choice (IIIG7) was transfected with a retroviral construct containing the green-fluorescence protein (GFP). The recipient dog was totally irradiated with 300 cGy and received a stem cell transplant with GFP-containing, immortalized, fibroblast-like monoclonal autologous stem cells (0.5 x 108/kg dog). No additional growth factors were applied. The peripheral blood counts recovered after 23 days (WBC >500; platelets >10,000). A peripheral blood smear showed some dim but definite, although timely, limited expression of the GFP protein in nucleated peripheral blood cells just five weeks after transplantation. A bone marrow biopsy showed GFP-positive cells in the marrow cavity predominantly as "bone-lining cells."


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The transplantation of blood progenitor cells was introduced into clinical medicine for the treatment of hematological malignancies and bone marrow failure as soon as it was recognized that spleen- and marrow-derived hematopoietic cells were able to reconstitute hematopoiesis. This therapeutic option became available after it could be shown that mice given myeloablative doses of irradiation did not die from treatment when their spleens were shielded by lead [1, 2]. Experiments in animals had shown that a sufficient number of hematopoietic stem cells can rescue those animals from marrow aplasia when injected i.v. after high-dose chemotherapy and total body irradiation (TBI) [3]. Hematopoietic stem cells are unique cells with the capacity for self-renewal and differentiation into all hematopoietic lineages [4].

It is generally accepted that the hematopoietic stem cell (HSC) resides in the bone marrow after birth and can circulate in the peripheral blood, from which it can be isolated by various measures [5]. Maintaining its viability and pluripotency requires a close interaction between the pluripotent HSC and the marrow microenvironment. The marrow microenvironment apparently consists of a heterogeneous population of cells, although predominantly of fibroblast-like cells. Those fibroblast-like cells are considered the major source of growth factors which induce differentiation and proliferation of HSCs [6-8] by the autocrine or paracrine production and secretion of those factors. Both HSCs and stromal cells are mesoderm-derived; they are still considered to be two different entities, although there is some evidence that there might be a common progenitor cell in the marrow stroma and hematopoiesis. Singer et al. described adherent common precursors for stromal and hematopoietic cells [9, 10], while Huang and Terstappen suggested that a single fetal CD34+, DR–, CD38– stem cell can differentiate into stromal elements (fibroblast-like cells) and cells with hematopoietic characteristics [11], although they had to amend their conclusions [12]. We previously established a canine marrow-derived stromal cell line which grows adherent as CD34– fibroblast-like cells [13]. Those cells can differentiate spontaneously into nonadherent CD34+ colony-forming hematopoietic precursor cells, although they require cell-to-cell contact and various growth factors to maintain their viability and capability to differentiate and proliferate [14]. Some molecular events involved in the differentiation and proliferation of CD34– precursor cells are already well known. The ligand for the tyrosine-kinase receptor c-kit (c-kit ligand or "stem cell factor") induces the differentiation of CD34– HSCs toward committed CD34+ progenitor cells. During differentiation, HSCs are arrested in the G0/G1 phase of the cell cycle, which is mediated by the overexpression of the cyclin-dependent kinase inhibitor p27kip-1 [15]. On the contrary, interleukin 6 (IL-6) induces proliferation of CD34– HSCs and maintains the adherent growth characteristics of those fibroblast-like precursor cells. IL-6 is also capable of reversing the transition from fibroblast-like, adherent growing CD34– HSCs to more committed CD34+ HSCs, an ability we already took advantage of to generate CD34– adherent growing HSCs from human peripheral blood mononuclear cells (PBMNCs) [16]. Fibroblast-like cells can differentiate spontaneously or in the presence of stem cell factor (SCF) into CD34+ nonadherent cells.

Here, we attempted to isolate CD34– fibroblast-like stem cells from canine PBMNCs. Those cells should be cloned, expanded, and transplanted into their autologous recipient to verify the pluripotency and ability for hematopoietic engraftment. Also of importance is whether CD34– fibroblast-like stem cells can also serve as mesenchymal precursor cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Establishment of a Peripheral-Blood-Derived Fibroblast-Like Stem Cell Clone
PBMNCs from 20 ml of heparinized blood were isolated by centrifugation of a Ficoll-Hypaque gradient (800 x g) for 20 min, and cells were placed in a tissue culture flask (Corning Costar; Cambridge, MA; http://www.corning.com) in the presence of IL-6 (200 U/ml; Calbiochem; Bad Soden, Germany; http://www.calbiochem.com). The IL-6-containing medium was changed twice per week. After the establishment of an adherent growing fibroblast-like cell layer in a 25 cm2 flask, the cells were immortalized using an SV-40 large T antigen containing plasmid (pUCIN-I) and lipofection with Superfect (Qiagen; Hilden, Germany). Immortalized cells were cloned and subcloned by limiting dilution as described previously [13]. Twelve clones were expanded in tissue culture flasks, and an aliquot of each cell clone was frozen and stored in liquid nitrogen.

GFP Transfection
One fibroblast-like clone named IIIG7 was expanded in tissue culture flasks. These fibroblast-like stem cells were infected with retroviral supernatant from the packaging cell line PG13, which were transfected with a retroviral vector containing the green fluorescence protein (GFP) (generously provided by Dr. D. Kohn, Children's Hospital; Los Angeles, CA). Retroviral supernatant of PG13 was used on fibroblast-like stem cells in the presence of IL-6. Infection with retroviral supernatant was repeated four times over a period of two weeks. Up to 95% of the retrovirally infected cells showed green fluorescence after four weeks.

Southern Blotting
IIIG7-GFP cells were grown to confluency in 75 cm2 flasks (NUNC; Wiesbaden, Germany; http://www.nalgenunc.com). Cells were harvested by trypsinization, and DNA was isolated using the "Qiagen Blood & Cell Culture DNA Midi Kit" (Qiagen). Purified DNA was digested with restriction enzymes, electrophoresed on 0.8% agarose gels and blotted on positively charged nylon membranes (Boehringer Mannheim; Manneheim, Germany). Blots were hybridized with a digoxigenin-labeled riboprobe, coding for the SV40 large T antigen. Detection of hybridization was performed by enzyme immunoassays (all solutions for labeling and detection of nucleic acids by Boehringer Mannheim).

Amplification of Canine

CD34 by RT-PCR   Total cellular RNA (1 µg) isolated from IIIG7-GFP cells was reverse-transcribed by MoMuLV reverse transcriptase ([RT]; Promega; Mannheim, Germany; http//ww.promega.com) in a 10-µl RT-assay (1 mM dNTPs, 1xRT-buffer, 100 ng oligo(dT), 10 U RNasin, 100 U M-MuLV-RT) for 1 h at 42°C. Polymerase chain reactions were done in a 50-µl reaction volume with 10 µl RT-mix, 10 pmol of each primer (CD34 sense: 5'-TGAGACCTCCAGCTGTGA-3', CD34 antisense: 5'-CAGGTGTTGTCTTGCTGAATGG-3'), and 1.5 U Taq polymerase (Boehringer Mannheim). cDNA was amplified for 40 cycles (50°C annealing temperature) followed by a seven-minute extension at 72°C after the last cycle in a thermocycler (Biozym; Hess, Germany). Amplified DNA fragments were separated on a 1.2% agarose gel and photographed after ethidium bromide staining.

Immunophenotyping for Canine CD34 and Osteocalcin
The monoclonal fibroblast-like stem cell clone IIIG7 was removed from the tissue culture flask by using a cell scraper and cell strainer (Becton Dickinson; http://www.bd.com) or enzymatic treatment. Cells were washed three times in phosphate buffered saline, counted, and adjusted to the appropriate concentrations: 106 cells were used per sample and were stained with polyclonal and monoclonal antibodies directed against canine CD34 (generously provided by Dr. Peter A. McSweeney, Fred Hutchinson Cancer Research Center; Seattle, WA) [17]. Specific binding was detected using a secondary fluorescein isothiocyanate-labeled antibody.

Fibroblast-like IIIG7 cells were further grown on chamber slides (NUNC) and also stained with the polyclonal and monoclonal antibodies directed against canine CD34. IIIG7 cells were further immunostained with the antibody directed against osteocalcin (BioTrend; Köln, Germany; http://www.biotrend.com). Cells were pretreated in a microwave oven for 15 min with the target retrieval solution (DAKO; Hamburg, Germany; http://www.dako.dk). Unspecific staining was blocked with a serum-free protein-block (DAKO). Cells were stained with the primary antibody for 60 min at room temperature and detected with the EnVision-labeled secondary AP-conjugated antibody (DAKO). Cells were counterstained with hemalum.

Clonal Assay for Colony-Forming Units (CFUs)
Adherent and nonadherent cells were removed from cultures, placed in a standard CFU assay (5 x 104 cells per dish), observed for colony formation in methylcellulose as described by Dexter et al. [18], and modified for canine cells by Schuening et al. [19]. IIIG7 cells were also incubated with SCF (50 ng/ml) and G-CSF (200 ng/ml) for seven days prior to the CFU assay.

Autologous Stem Cell Transplantation
Fibroblast-like stem cell clones were isolated from a 16-month-old male Beagle, which was irradiated with 300 cGy (70 cGy/min) TBI. The dog was transplanted with 0.5 x 108 cells/kg of the fibroblast-like stem cell clone IIIG7 by injecting the cells slowly into a foreleg vein. Three hours were required to isolate 8 x 108 cells in total from tissue culture flasks and infuse these cells i.v. The dog showed no adverse reaction, either throughout the procedure or afterward. Both the radiation regimen and cell dose corresponded to those used previously and published by others [20].

Peripheral Blood Cell Analysis
Blood was obtained on a daily basis post-transplant and immediately transferred into a heparin-containing vial. Cell counts were obtained by means of an automated counter. GFP fluorescence was determined by fluorescence microscopy using a UV-light microscope (Zeiss Axiophot; Jena, Germany).

Bone Marrow Examination
A bone marrow biopsy was taken from the humerus of the transplanted dog 9 and 15 weeks after transplantation. The biopsy was fixed immediately overnight in buffered 4% formalin and decalcified for 3 h in EDTA. After a 10 min rinse in water, the bone was further fixed, processed with alcohol and melted paraffin at 56°C, and finally, embedded. Microtome sections were stained with hematoxylin and eosin according to standard procedure. Sections were further evaluated for green fluorescence in marrow cells using a UV-light microscope (Zeiss Axiophot).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Monoclonal, Fibroblast-Like, Peripheral-Blood-Derived Stem Cell Line
Peripheral-blood-derived mononuclear cells started to grow adherently in IL-6-containing medium 10-14 days after culture initiation. The initial adherent growing cells also produced matrix proteins, such as fibronectin (unpublished). After the adherent growing cells were immortalized using the SV40 large T antigen, they reached confluency in culture after three to five weeks. The fibroblast-like cells were then successfully cloned and subcloned as described previously [13], and various cell clones were propagated. The clones show the typical fibroblast-like morphology, although some cells started to detach and remain in culture as nonadherent round cells (Fig. 1Go), a phenomenon we have already described as spontaneous "in vitro" differentiation [14]. One clone, named IIIG7, was further expanded and transfected using a GFP construct. Ninety-five percent of the transfected cells showed a definite but dim stain for green fluorescence.



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  		Figure 1. Typical fibroblast-like morphology of the adherent growing canine stem cell clone IIIG7. This cell clone was derived from peripheral blood mononuclear cells after Ficoll-gradient separation, immortalization with SV-40 T antigen, and single-cell cloning by limiting dilution. A small number of cells detach from the plastic surface (~3%) and remain in culture as nonadherent, small lymphocyte-like cells. These nonadherent cells can reattach to the plastic surface of the tissue culture flask after interacting with other still-adherent growing fibroblast-like cells.

 
Colony Formation of the Fibroblast-Like Cell Line IIIG7-GFP
As already described for the canine-marrow-derived CD34– stem cell line D064 [13], we showed colony-formation potential of the peripheral blood-derived, fibroblast-like clone IIIG7 in a standard CFU assay. Since the predominantly adherent growing cells can spontaneously differentiate in culture (Fig. 1Go), we distinguished between adherent growing and nonadherent cells in culture. Both cell types were able to produce cell clusters and CFUs (Fig. 2Go), although there was a significant difference between adherent and nonadherent cells (Table 1Go). Adherent cells showed a higher CFU potential than the nonadherent cells, especially if treated in culture with additional growth factors (SCF/G-CSF) prior to the colony assay.



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  		Figure 2. Colony-forming potential of adherent growing and nonadherent IIIG7 cells. Adherent and nonadherent cells were used in a CFU assay in methylcellulose [13]. The cells were stimulated with SCF (50 ng/ml) and G-CSF (200 ng/ml) for seven days prior to the CFU assay (Table 1Go). The figure shows a colony from an adherent cell culture.

 

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  		Table 1. Colony formation of the IIIG7 clone after 14 days in a standard assay. Some fibroblast-like stem cells were treated in culture with SCF and G-CSF for seven days prior to placement in methylcellulose.
 
Clonality
DNA was extracted from IIIG7 cells and digested with restriction enzymes. The fragments were electrophoresed and blotted on nylon membranes, which were hybridized with an SV40 large T-antigen-specific probe. Only one single and specific band per digest was detectable, showing that IIIG7 cells are of monoclonal origin (not shown).

CD34 Expression and CD34 mRNA Transcription in the Peripheral-Blood-Derived Stem Cell Line
Immunophenotyping of IIIG7 cells showed a heterogenous population with regard to CD34 expression (Fig. 3AGo). The CD34– canine-marrow-derived hematopoietic stem cell line D064 served as a negative control (top) [13], while IIIG7-GFP cells already had a broad baseline fluorescence without any primary antibody against CD34 (middle). IIIG7-GFP cells showed a broad CD34– population and a single, sharp peak for low CD34-expressing cells (bottom). In contrast to the marrow-derived stem cell line D064, which was completely CD34–, the peripheral-blood-derived and GFP-transfected stem cell line IIIG7 obviously contained CD34– as well as CD34low-expressing cells, suggesting a dynamic shift between CD34 negativity and CD34 expression. Endothelial cells were used as control cells, and they showed a high CD34 expression (not shown). This expression pattern is confirmed in the mRNA transcription of CD34 (Fig. 3BGo). IIIG7-GFP cells transcribe CD34 (arrow), while all other marrow-derived cell lines (D064 and DR+ D064) do not transcribe any message for CD34 [13, 14, 21, 22]. Also, immunohistochemistry with the anti-canine CD34 monoclonal antibody shows the coexistence of CD34– and CD34low-expressing cells in the tissue culture (Fig. 3CGo). It appears to the experienced eye that the CD34– cells are flat on the bottom of the tissue culture flask surface, while CD34low cells grow on top of them.





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  		Figure 3. CD34 expression in peripheral-blood-derived fibroblast-like stem cells. A) The CD34– canine-marrow-derived hematopoietic stem cell line D064 served as a negative control (top) [13, 14], while IIIG7-GFP cells already had a broad baseline fluorescence without any primary antibody against CD34 (middle). IIIG7-GFP cells show a broad CD34– population and a single, sharp peak for low CD34 expressing cells (bottom). B) IIIG7-GFP cells transcribe predominantly the full-length form of CD34 (arrow) but also the truncated form, while all other marrow-derived cell lines (D064 and DR+ D064) do not transcribe any message for CD34 [36]. C) Immunohistochemistry with the anti-canine CD34 monoclonal antibody shows the coexistence of CD34– and CD34low-expressing cells in the tissue culture. It appears to the experienced eye that the CD34– cells are flat on the bottom of the tissue culture flask surface, while CD34low cells are on top of those.

 
Expression of Osteocalcin
Expression of osteocalcin in peripheral-blood-derived fibroblast-like cells is dependent upon cell culture conditions and cell density. With time in culture, IIIG7 cells tend to form complex cluster-like structures which start to express osteocalcin (Fig. 4Go). Fibroblast-like cells at low density do not express osteocalcin.



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  		Figure 4. Expression of osteocalcin in IIIG7 cells. IIIG7 cells start to express osteocalcin as a mesenchymal marker in cluster-like structures with a high cell density. Marginal fibroblast-like cells do not express osteocalcin.

 
Engraftment of CD34– Monoclonal Cells
The CD34–/low fibroblast-like stem cell line IIIG7 was derived from canine peripheral blood cells. The donor dog received a TBI with 300 cGy and an autologous stem cell transplant with GFP-transfected IIIG7 cells (0.5 x 108 cells/kg). The radiation dose was chosen on the basis of chimeric allogeneic transplantation models to achieve at least a possible chimerism of transplanted and endogenous hematopoietic cells. We used a high cell transplant dose to certify engraftment in the event that IIIG7 cells are true hematopoietic stem cells. Figure 5Go shows the peripheral white blood cell and platelet count recovery in the dog after radiation and stem cell transplant. A bone marrow biopsy five weeks after transplantation already shows GFP-expressing cells predominantly along the bone spicules ("bone lining cells") and very few possible myeloid progenitors with a green fluorescence (Fig. 6Go). These "bone lining cells" also showed expression of osteocalcin (Fig. 7Go). Although some green fluorescence of very few nucleated peripheral blood cells was observed at some points in time (weeks 3 and 5 post-transplant), there is still evidence that the engrafted IIIG7-GFP clone contributed long-term to the hematopoietic engraftment.



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  		Figure 5. Reconstitution of the autologously transplanted dog. Peripheral white blood cell and platelet counts after autologous transplantation of our Beagle dog with the autologous peripheral-blood-derived monoclonal stem cell line IIIG7. After TBI with 300 cGy, the dog recovered with counts at about day 23 (WBC >500/µ and platelets >50,000/µl) without additional growth factor treatment. The blood smears showed a normal differentiation.

 


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  		Figure 6. Bone marrow engraftment of GFP-IIIG7 cells. Bone marrow biopsy of the dog transplanted with the autologous stem cell clone IIIG7 five weeks after transplantation. At this time point after transplantation, it is the "bone lining cells" that predominantly express the GFP protein, suggesting that this is the privileged homing site of the CD34–/low, fibroblast-like hematopoietic stem cells.

 


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  		Figure 7. Osteocalcin expression of bone lining cells. The bone marrow biopsy at week 5 also shows osteocalcin expression in periosteal cells (red), which represent the same location as the GFP+ cells (compare Fig. 6Go).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD34– fibroblast-like stem cells can be isolated from human peripheral blood mononuclear cells in primary cultures in the presence of IL-6 [16]. Here, we isolated CD34–/low hematopoietic stem cells from a small amount of peripheral blood from one dog. This dog was engrafted with autologous, in vitro expanded, cloned, fibroblast-like stem cells and showed trilinear hematopoietic recovery. The appearance of GFP-expressing nucleated cells in the periphery showed a cycling rhythm, a phenomenon which was previously described for gene marking studies [23]. Also, Osawa et al. could already show that lymphohematopoietic reconstitution is possible by a single CD34–/low hematopoietic stem cell [24], which was confirmed by others more recently [25, 26]. Here we used a peripheral-blood-derived monoclonal cell line to achieve engraftment in the bone marrow. This discrepancy can be explained if myeloid and megakaryocytic differentiation occurs earlier than lymphoid differentiation, during hematopoietic differentiation. The CD34–/low fibroblast-like cell line IIIG7 as well as the marrow-derived CD34– cell line D064 [13] might represent the earliest stage of hematopoietic progeny, capable of differentiating into cells with mesenchymal characteristics.

Previous experiments with canine, murine, and human fibroblast-like stem cells had already demonstrated that single-cell cloning was impossible without at least temporary immortalization. This is due to a low precursor frequency of those circulating hematopoietic stem cells in the small amount of peripheral blood used in our approach. To maintain viability and retain the ability to proliferate and differentiate, the CD34– fibroblast-like stem cells need cell-to-cell contact and a microenvironment which provides necessary growth factors in adequate quantity. All those factors are produced by those fibroblast-like stem cells in an autocrine or paracrine fashion [16]. However, the number of stem cells circulating in the peripheral blood and capable of returning to a state of CD34– adherent growth is too small to provide a self-established viability-maintaining microenvironment in vitro.

The precursors of our CD34–/low fibroblast-like stem cell in the peripheral blood are probably among the CD34+ population. Although a positive selection of CD34+ cells did not increase the yield of colony formation of CD34– stem cells (authors' unpublished experiments), it is most likely that CD34+ circulating stem cells home to the marrow cavity, where they rest as fibroblast-like CD34– stem cells until differentiation can occur again. We have called this event the "stem cell cycle" [27]. When CD34+ circulating stem cells return to a state of adherent growth, p27kip-1 accumulates in the cell [15], leading to an arrest of the majority of cells in G0/G1 phase of the cell cycle. This "quiescence" of the majority of cells, which apparently depends on IL-6 for proliferation [14], is also due to a lack of STAT activation and a downregulation of cdc2 (Huss et al., in press). In this way, a large number of adherent growing CD34– cells are temporarily refractory to respond to a proliferation signal. The mechanisms for determining what cell is refractory for a certain period of time are still unknown, but seem to be very efficient for providing a sufficient pool of hematopoietic stem cells for the span of a lifetime.

Colony formation was observed especially in fibroblast-like stem cells when pretreated with early-acting hematopoietic growth factors such as SCF and G-CSF, suggesting that CD34–/low fibroblast-like stem cells proliferate in culture in the presence of autocrine or paracrine acting growth factors, such as IL-6 [14].

As demonstrated here and elsewhere, there seems to be a difference between marrow-derived CD34– and peripheral-blood-derived CD34–/low fibroblast-like stem cells. Although both cell lines, D064 and IIIG7, produce CFUs and even long-term culture-initiating cells [13], there might be a difference in the state of activation. Since peripheral-blood-derived IIIG7 cells tend to express CD34 earlier than marrow-derived D064 cells, stem cells which participate in the "stem cell cycle" are at a higher state of activation than predominantly resting and quiescent stem cells. Nevertheless, there is certainly a permeability of this system depending on the cellular demand in the periphery during infection, blood loss, etc., allowing "quiescent" stem cells to become "active" and "active" stem cells to start to circulate and eventually terminally differentiate (Fig. 8Go).



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  		Figure 8. Schematic presentation showing how peripheral-blood-derived hematopoietic stem cells home to the marrow cavity. The majority of cells rest as CD34–/low "bone lining" cells along the bone spicules as "quiescent stem cells," while some CD34+/low "active stem cells" start to terminally differentiate or return to circulation in the peripheral blood. This is called the "stem cell cycle" [27].

 
Trying to identify the most immature hematopoietic stem cell population, we should not forget that we can only take a glance at a dynamic system in vitro, which is dependent on a probably uncountable number of factors in a very complex eukaryotic organism.

In view of our results, the observations by Anklesaria et al. [28] and Keating et al. [29] might have to be reinterpreted in that they have not necessarily transplanted bone marrow stromal cells but possibly fibroblast-like stem cells resting within the marrow microenvironment. Our results on the intermittent GFP expression further represent some, although preliminary, evidence of the oscillatory nature of hematopoiesis, suggesting the cycling of different stem cell populations between the bone marrow and the peripheral blood [30]. We further demonstrated that the CD34– fibroblast-like stem cell clone IIIG7 as well as the "bone lining cells" also expressed osteocalcin as a marker for mesenchymal progenitors and osteogenetic precursors. In view of recent publications [31, 32], it is conceivable that this is a common precursor cell of hematopoiesis and generating mesenchymal stem cells giving rise to bone, cartilage, and other mesenchymal organ systems. "Bone lining cells" are usually interpreted as osteoblasts which give rise to the bone structure in the marrow cavity. But our own previous results showed that these osteoblast-like cells were also transplanted by a peripheral-blood-derived CD34– stem cell clone in mice [33], which also expressed c-kit as a marker of early hematopoietic progeny.

We are apparently only beginning to comprehend the enormous potential of CD34– hematopoietic and mesenchymal stem cells, as they can be generated from various sources and activated by various measures [34, 35].


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors would like to thank Stephanie Theis, Sabine Sagebiel-Kohler, Karin Oettrich, and Sabine Moosmann for their excellent technical assistance, and Monika Franz, D.V.M, Günther Merx, D.V.M., and Michael Hagemann for their animal care.

Supported in part by a grant of the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany and a Fellowship of the International José Carreras Leukemia Foundation, Barcelona, Spain (to R.H.).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received April 27, 2000; accepted for publication May 1, 2000.




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