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Stem Cells 2003;21:98-104 www.StemCells.com
© 2003 AlphaMed Press

RAPID COMMUNICATION

Liver-Specific Gene Expression in Cultured Human Hematopoietic Stem Cells

Henning C. Fiegela,b, Michael V. Lioznova, Lourdes Cortes-Dericksa, Claudia Langea, Dietrich Kluthb, Boris Fehsea, Axel R. Zandera

a Bone Marrow Transplantation Center and
b Department of Pediatric Surgery, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany

Key Words. Hepatic stem cells • Adult bone marrow stem cells • Stem cell culture

Henning Fiegel, M.D., Department of Pediatric Surgery, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. Telephone: 49-40-42803-3494/6918; Fax: 49-40-42803-6914; e-mail: fiegel{at}uke.uni-hamburg.de


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. Based on the recent observations that hepatocytes may originate from bone marrow, we investigated the potential of CD34+ bone marrow cells to differentiate into hepatocytic cells in vitro. CD34+ and CD34- human bone marrow cells were separated by magnetic cell sorting. Cells were cultured on a collagen matrix in a defined medium containing hepatocyte growth factor. Cell count and size were measured by flow cytometry, and reverse transcription polymerase chain reaction was carried out for the liver-specific markers CK-19 and albumin. During cell culture, CD34+ cells showed an increasing cell number and proliferative activity as assessed by Ki-67 staining. Under the specified culture conditions, CD34+ cells expressed albumin RNA and CK-19 RNA after 28 days, whereas CD34- cells did not show liver-specific gene expression. The results indicate that CD34+ adult human bone marrow stem cells can differentiate into hepatocytic cells in vitro.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Liver cells originating from bone marrow were first observed in patients after bone marrow transplantation for hematological disorders. In autopsies, differentiated donor-derived hepatocytes were identified in the recipient livers by immunohistochemical analysis [1]. Also, bile duct epithelial cells were found to be derived from bone marrow [2]. Recently, similar findings were described after the transplantation of mobilized peripheral blood stem cells [3]. Animal studies revealed that hepatocytes and liver stem cells (oval cells [OCs]) may originate from bone marrow under certain conditions. Initially, Petersen et al. showed in rats that bone marrow cells could differentiate into OCs after combined liver damage and bone marrow transplantation [4]. Furthermore, Theise et al. found a differentiation of bone marrow cells into liver cells in mice without liver damage [5].

Tissue-residing stem cells of the liver were identified in adult rodent and human livers by coexpression of stem cell markers (e.g., CD34, Thy1, and c-kit) and hepatocytic lineage markers (CK-18, CK-19, alpha fetoprotein, and albumin) [6, 7]. These cells have been described as OCs. It was shown that OCs can differentiate bipotentially either into hepatocytes or into bile duct epithelial cells [8], and they have clonogenic potential in vivo [9] and in vitro [10]. Isolation of such cells from human livers was achieved recently, and their differentiation into biliary epithelial duct-like cells in vitro has been shown [11]. Normally, OCs are not involved in liver regeneration since mature hepatocytes have an enormous proliferative potential [12]. Only when parenchymal hepatocytes are damaged, or their proliferative capacity is blocked, does the recruitment of OCs take place [13].

The potential of adult bone marrow stem cells to differentiate into nonhematopoietic tissues (e.g., endothelial cells [14], skeletal muscle [15], neuronal cell types [16, 17]) was revealed in the past years by several groups. In mouse studies, multiorgan and multilineage engraftment of single bone marrow-derived stem cells was demonstrated [18]. Also in humans, engraftment of peripheral blood stem cells was observed in several tissues (gut, skin, liver) [3]. These findings led to the assumption that there exist stem cells (e.g., mesenchymal stem cells, multipotent adult progenitor cells [MAPCs]) in the bone marrow that possess multilineage differentiation capacity [19, 20]. A recent study demonstrated that MAPCs can differentiate into adult tissues (e.g., into hepatocytic cells) in vitro [21]. The ability of hematopoietic stem cells to differentiate into nonhematopoietic tissues, e.g., endothelial cells (hemangioblasts), is currently suggested [22]. Lagasse and coworkers achieved a repopulation of chronically diseased livers by healthy, sorted c-kit-positive mouse bone marrow stem cells (KTLS cells) in enzyme-deficient mice (fumarylacetoacetate hydrolase deficient) [23]. Since such hematopoietic stem cells and the previously described hepatic OCs share the same well-known stem cell markers (CD34, Thy1), the question of a functional relationship—in the meaning of a stem cell hierarchy—is challenging. Therefore, in this study we investigated the potential of sorted adult human bone marrow stem cells to differentiate into hepatic-lineage cells in vitro.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Cell Isolation and Characterization
Human bone marrow was harvested from healthy adult donors for allogeneic bone marrow transplantation. Human material was used after informed consent of the donor and according to the guidelines from the ethical committee of the Ärztekammer Hamburg (OB/V/02). Mononuclear cells were obtained by a Ficoll-separation step. CD34+ bone marrow cells were separated by magnetic cell sorting (MACS), using the human CD34-progenitor kit (Miltenyi Biotec; Bergisch-Gladbach, Germany; http://www.miltenyibiotec.com) according to the manufacturer’s instructions. The flow-through fraction was used as a source of CD34- cells. Fluorescenceactivated cell sorting (FACS) analysis with anti-CD34-phycoerythrin (PE) (Beckton Dickinson; San Diego, CA; http://www.bd.com), anti-CD45-PE, anti-CDw90-fluorescein isothiocyanate, and anti-CD117-PE (all Immunotech; Hamburg, Germany) was performed on an Epics XL-MCL FACS analyzer (Beckman Coulter; Krefeld, Germany; http://www.beckman.com) to characterize the isolated cells. To assess the unspecific binding, appropriate isotype controls were included.

Groups and Culture Conditions
In culture, either CD34+ sorted cells or CD34-depleted cells from bone marrow were examined. Cultures were analyzed on days 0, 4, 7, 14, 21, 28, and 35. Cells were seeded on collagen type I (Sigma; St. Louis, MO; http://www.sigmaaldrich.com) coated 96-well plates (Nunclon multidishes) at a concentration of 2.0 x 106 cells/ml in a defined culture medium. For the medium, 500 ml Williams Medium E without L-glutamine (GIBCO BRL; Eggenstein, Germany; http://www.lifetech.com) was supplemented with 50 mg/l L-glutamine (GIBCO), 100 IU/l penicillin/streptomycin (Sigma), 20 mM HEPES, 20 mM sodium pyruvate (GIBCO), 5 nM dexamethasone (Sigma), 10 ng/ml epidermal growth factor (GIBCO), 5 ng/ml hepatocyte growth factor (HGF; Sigma), 20 mU/ml insulin (GIBCO), 10% fetal calf serum (Sigma), and 10% horse serum. Medium was changed every other day.

Flow Cytometry and Statistical Analysis of Cell Number and Volume
Flow cytometry was done with freshly isolated or cultured cells. Cell number and cell volume in femtoliters (fl = 10-15 liter) were assessed by the MicroDiff 18 analyzer (Beckmann Coulter). Mean values and standard deviations (SD) of cell number and volume were calculated. Statistical analysis were performed employing the student t-test on Microsoft® Windows® using Excel 2000 software (Microsoft; Redmont, WA; http://www.microsoft.com). p values were two sided and p < 0.05 was considered significant.

Cytospins and Immunocytochemistry
Cells were harvested from cultures by washing with phosphate-buffered saline (PBS). Cytospins were prepared by centrifugation of the cell suspension (400 x g for 10 minutes) on glass slides. Cytospins were fixed with methanol at -20°C for 5 minutes and acetone at 4°C for 15 seconds. Immunohistochemical analysis for Ki-67 was performed using the alkaline phosphatase-antialkaline phosphatase (APAAP) technique using mouse monoclonal antibodies (mAbs) (Pharmingen; Hamburg, Germany; http://www.bdbiosciences.com/pharmingen). The incubation period with primary antibody (diluted 1:50) was 30 minutes. Secondary marking was done with rabbit-anti-mouse IgG mAb (1:50) for 30 minutes. Slides were then incubated with mouse-APAAP complex for 30 minutes. The alkaline phosphate substrate, New Fuchsin, was prepared as described elsewhere [24]. Slides were counterstained with hematoxylin.

RNA Extraction from Cultured Cells
RNA was isolated from cultured cells and liver tissue after rinsing the cells with PBS. 105-106 cells were homogenized in 200 µl RNAzol (Wak Chemie; Bad Homburg, Germany; containing guanidine thiocyanate and mercaptoethanol) and were kept on ice. For extraction, 200 µl chloroform was added, and the mixture was incubated for 5 minutes at -20°C. After centrifugation at 6,000 rpm for 15 minutes, the supernatant was mixed with 500 µl isopropanolol-2 and 2.5 µl glycogene, and the mixture was incubated at -20°C for 30 minutes. After centrifugation at 6,000 rpm for 30 minutes, the supernatant was removed and 750 µl ethanol was added for precipitation. This step was repeated twice before the precipitates were vacuum dried and solved in 50-70 µl diethyl pyrocarbonate (DEPC) water. The OD260/OD280 ratio was measured using photometry (Uvikon) to determine the RNA content. One microgram of RNA was dissolved in a total volume of 8 µl DEPC water and stored for reverse transcription (RT) at -80°C.

RT-Polymerase Chain Reaction (RT-PCR)
RT of extracted RNA was performed using the First-strand c-DNA synthesis kit (Amersham Bioscience Europe; Freiburg, Germany; http://www.apbiotech.com) according to the manufacturer’s instructions. Briefly, RNA was denaturated for 10 minutes at 65°C. Then, bulk mix (containing RT 10 x PCR buffer and MgCl2), dithiothreitol, and PdN6 primer were added to a total volume of 15 µl. The RT reaction was allowed to proceed at 37°C for 60 minutes. The cDNA was stored at -20°C.

PCR with cDNA was performed using the following primers: 5'-CCT TCA TTG ACC TCA ACT AC-3' and 3'-GGA AGG CCA TGC CAG TGA GC-5' for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 5'-TTA GGA ATC CCC CAG GAA GAC ATC CTT TGC-3' and 3'-CCT GAG CCA GAG ATT TCC-5' for albumin [25]. For CK-19 PCRs, the primers were 5'-TTT GAG ACG GAA CAG GCT CT-3' and 3'-CAG CTC AAT CTC AAG ACC CTG-5', and the nested primers were 5'-GCA GAT CGA AGG CCT GAA-3' and 3'-TGA ACC AGG CTT CAG CAT C-5' [26]. For the PCR reaction, 7 µl cDNA template was mixed with 5 µl 10 x PCR buffer, 1 µl 10 mM deoxyribonucleoide 5' triphosphate, 1.5 µl 50 mM MgCl2, 1 µl primers (50 ng/µl), and 1 µl polymerase (Ampli-Taq; GIBCO) for each probe. PCR was carried out in a programmable Uno-Thermobloc (Biometra; Göttingen, Germany; http://www.biometra.de) with the following conditions: 94°C for 10 minutes and then 30 (GAPDH or CK-19 I/II) or 35 (albumin) cycles, each comprising denaturation for 1 minute at 94°C; annealing for 1 minute at 62°C for GAPDH, 55°C for albumin, or 58°C for CK-19 I/II; and then extension for 1 minute at 72°C. After PCR was completed, reaction tubes were kept for 5 minutes at 72°C and then 4°C. Negative controls routinely used for each set of primers included control without template. Samples were analyzed on 1.5% agarose gels. The size of the PCR fragments was estimated using a 100-bp ladder (GIBCO BRL).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Characterization of Isolated CD34+ Bone Marrow Stem Cells
Bone marrow was harvested from 34 healthy donors for allogeneic bone marrow transplantation. Male-female ratio was 25/9 (73.5% male), and the mean donor age was 36.9 ± 11.4 years. Per isolation, the yield was 2.0 ± 1.4 x 106 for CD34+ cells and 1.4 ± 0.4 x 108 for CD34- cells. FACS analysis of positive sorted cells revealed that after MACS, 93.5% ± 2.6% of cells were positive for CD34, whereas in the depleted fraction, 0.10% ± 0.08% CD34+ cells were seen. Of the CD34+ fraction, 97.7% was positive for CD45 and 15.7% was positive for CD117.

Cell Number and Morphology During Culture Period
Culture of CD34+ cells showed increasing cell numbers (Table 1AGo), with a significant (p < 0.005) rise in cell count from 258.3 ± 13.29 x 103 cells/well at day 0 to 355.0 ± 5.77 x 103 cells/well at day 35 in culture (Fig. 1Go). A peak in cell growth (p < 0.01) was observed between day 21 (230.9 ± 102.12 x 103 cells/well) and day 28 (403.8 ± 116.16 x 103 cells/well). Cell size increased significantly (p < 0.05) from 75.6 ± 8.73 fl at day 0 to 129.6 ± 7.76 fl at day 21 (Table 1BGo). At day 28, two populations of cells were found in the cultures of CD34+ cells: a minority (<1%) of large cells with a volume of 110.0 ± 5.55 fl, and a majority of small cells with a volume of 40.0 ± 2.77 fl (Fig. 2Go). Number of cultured CD34- cells decreased significantly (p < 0.05) from 370.0 ± 60.33 x 103 cells/well at day 0 to 92.2 ± 16.41 x 103 cells/well at day 35. CD34- cell size was constant over the whole observation period from day 0 (50.0 ± 3.16 fl) until day 35 (48.8 ± 2.31 fl). Cell numbers of cultured CD34+ cells increased significantly (p < 0.05) after day 7 when compared with numbers of cultured CD34- cells (Table 1AGo and Fig. 1Go). Cell volume of CD34+ cells was significantly (p < 0.005) higher than volume of CD34- cells until day 21 (Fig. 2Go).


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  		Table 1. A) Cell numbers (1,000/well; mean ± SD) of cultured CD34+ and CD34- cells over time. p values show statistical significance between CD34+ and CD34- cells at each time point. B) Cell volume (in femtoliters; mean ± SD) of cultured CD34+ and CD34- cells over time. In cultures of CD34+ cells, two populations appeared at day 28: a majority of small cells and a minority (<1%) of large cells.
 


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  		Figure 1. Number of cultured CD34+({diamondsuit}) stem cells increased significantly (p< 0.005), with a peak in growth between day 21 and day 28 (*p< 0.01). After day 7, cell numbers of cultured CD34+cells were significantly (p < 0.05) higher than those of CD34-({bullet}) cells. Numbers of CD34-cells decreased (p < 0.05) over the whole observation period. Cell number is shown in 1,000 cells/well (mean value ± SD) of CD34+and CD34-human bone marrow cells cultured on collagen with addition of HGF (n = 9 each).

 


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  		Figure 2. Cell volume of cultured CD34+({diamondsuit}) stem cells increased until day 21 of culture (*p< 0.05). After day 28, two populations were observed in cultures of CD34+cells: a minority (<1% of all cells) of large cells ({blacksquare}) and a majority of smaller cells ({blacktriangleup}). CD34-cells ({bullet}) were significantly smaller (p < 0.005) than CD34+cells until day 21 and showed a constant cell volume over the whole observation period. Cell volume in femtoliters (mean values ± SD) as assessed by flow cytometry (n = 7 each).

 
Ki-67 Immunocytochemical Staining
Ki-67+ cells were observed at all time points in cultures of CD34+ cells (Fig. 3Go), but a decrease of positivity for Ki-67 was observed over the whole observation period. CD34- cells showed no Ki-67+ cells (data not shown).



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  		Figure 3. Immunocytochemical staining for Ki-67 showed active proliferation (arrows) of CD34+cells cultured under hepatocyte-specific conditions at day 14 (APAAP, magnification x40).

 
RT-PCR for Albumin
RT-PCR analysis for albumin mRNA resulted in no signal in cultured CD34+ bone marrow stem cells before day 28 (data not shown). After day 28, cultured CD34+ cells became positive for albumin mRNA as detected by RT-PCR (Fig. 4Go), and at the same time became positive for CK-19 mRNA. RT-PCR analysis of human liver tissue confirmed signals for albumin-cDNA at 250 bp, and CK-19-cDNA at 190 bp. Cultures of CD34- bone marrow cells showed no signal for albumin-mRNA at all time points (Table 2Go). Unstable and rare CK-19 mRNA expression was observed in cultures of CD34- bone marrow cells.



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  		Figure 4. RT-PCR gel image analysis of CD34+human bone marrow stem cells cultured under hepatocyte-specific conditions with high-dose HGF and collagen type-I matrix for 35 days. Arrows indicate positive signals for gene expression of hepatocytic marker CK-19 (++) at 190 bp (lane 1) and albumin (+) at 250 bp (lane 2).

 

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  		Table 2. Summary of RT-PCR analysis of cultured CD34+ and CD34- human bone marrow cells under hepatocyte-specific culture conditions
 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
In this study, CD34+ hematopoietic stem cells were isolated by MACS from adult human bone marrow. The hypothesis that CD34+ stem cells may differentiate into hepatic cells was based on two reasons: A) Tissue resident liver stem cells (OCs) also bear hematopoietic stem cell markers, such as CD34 or Thy-1 [4, 7, 13], and B) bone marrow, containing the most well-known CD34+ stem cells, can differentiate into hepatocyte-like cells in vivo [1–5].

The influence of environmental factors (e.g., cytokines, extracellular matrix components) on cellular differentiation is well known, e.g., for the culture of adult hepatocytes. In our study, hepatocyte-specific conditions were used to induce hepatic-cell-like differentiation of the cultured bone marrow cells. Our data show a significant increase in the number of CD34+ cells under the specified cell culture conditions. Ki-67 staining revealed that proliferation of cultured CD34+ cells was seen at all time points. PCR analysis for the liver cell-specific genes albumin and CK-19 indicate that CD34+ bone marrow cells began to express liver-specific genes after 28 days in culture. This may be explained as a result of gradual differentiation into a hepatic-stem-like cell in vitro. Oh et al. found similar results in cultures of total bone marrow from the rat, which expressed liver-specific genes and proteins after a 3-week period [27]. In that study, the impact of environmental factors is highlighted because a hepatic differentiation of the cultured cells was observed only in cultures with high-dose HGF [27]. Schwartz et al. investigated the capacity of human nonhematopoietic bone marrow stem cells (MAPCs) to differentiate into liver-cell-like cells in vitro [21]. They showed that human MAPCs can express liver-specific genes when cultured under hepatocyte-specific conditions. These studies, together with the previously mentioned in vivo studies [1–5, 23], strongly suggest that the idea of a potential "extrahepatic" liver stem cell residing in the bone marrow does exist. Our study supports this assumption. We have shown in vitro for the first time that human adult hematopoietic stem cells also have an enhanced differentiation capacity. Following stem cell culture under specified conditions, cells with liver-cell-specific characteristics were found.

Hepatic stem cells may become important for the development of new therapeutic strategies for liver diseases [28]. Experimental treatment of liver diseases by repopulation of diseased livers with healthy hepatocytes [24] or hepatocyte transplantation [29], tissue engineering of the liver [30], and ex vivo gene therapy have achieved promising results in animal models [31]. For such approaches, bone-marrow-derived stem cells provide several advantages over hepatocytes: A) Bone marrow stem cells are easily obtained; B) transduction of stem cells may result in the expansion of "cured" daughter cells [32]; C) bone marrow and stem cell transplantation is clinically well established [33], and D) regarding ethical issues, the use of adult stem cells is favorable over other stem cells, such as embryonic stem cells or fetal stem cells. Thus, the potential of adult bone marrow stem cells to differentiate into functional hepatocyte-like cells could be of high interest for new cell-based therapies.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Our in vitro data indicate that human hematopoietic stem cells may be potential liver stem cells. Therefore, the use of such cells for new cell-based therapies for liver diseases is worth studying. Further studies must define the in vivo potential and conditions for differentiation of bone marrow into liver.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
We want to thank PD Dr. W. H. Kruger, Department of Hematology and Oncology, University Hospital Halle, Germany, for kindly providing the primers for our CK-19 PCR analysis, and Mrs. T. Gröger and Mrs. M. Appl for their technical assistance.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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Received September 4, 2002; accepted for publication October 8, 2002.



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STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS