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First published online June 15, 2006
Stem Cells Vol. 24 No. 10 October 2006, pp. 2244 -2251
doi:10.1634/stemcells.2006-0141; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Properties of Cryopreserved Fetal Liver Stem/Progenitor Cells That Exhibit Long-Term Repopulation of the Normal Rat Liver

Michael Oertel, Anuradha Menthena, Yuan-Qing Chen, David A. Shafritz

Marion Bessin Liver Research Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, USA

Key Words. Fetal liver stem/progenitor cells • Cell transplantation • Cryopreservation • Liver repopulation

Correspondence: David A. Shafritz, M.D., Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. Telephone: 718-430-2098; Fax: 718-430-8975; e-mail: shafritz{at}aecom.yu.edu

Received March 10, 2006; accepted for publication June 7, 2006.
First published online in STEM CELLS EXPRESS   June 15, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
We have previously achieved a high level of long-term liver replacement by transplanting freshly isolated embryonic day (ED) 14 rat fetal liver stem/progenitor cells (FLSPCs). However, for most clinical applications, it will be necessary to use cryopreserved cells that can effectively repopulate the host organ. In the present study, we report the growth and gene expression properties in culture of rat FLSPCs cryopreserved for up to 20 months and the ability of cryopreserved FLSPCs to repopulate the normal adult rat liver. After thawing and placement in culture, cryopreserved FLSPCs exhibited a high proliferation rate: 49.7% Ki-67-positive on day 1 and 34.7% Ki-67-positive on day 5. The majority of cells were also positive for both {alpha}-fetoprotein and cytokeratin-19 (potentially bipotent) on day 5. More than 80% of cultured cells expressed albumin, the asialoglycoprotein receptor, and UDP-glucuronosyltransferase (unique hepatocyte-specific functions). Expression of glucose-6-phosphatase, carbamyl phosphate synthetase 1, hepatocyte nuclear factor 4{alpha}, tyrosine aminotransferase, and oncostatin M receptor mRNAs was initially negative, but all were expressed on day 5 in culture. After transplantation into the normal adult rat liver, cryopreserved FLSPCs proliferated continuously, regenerated both hepatocytes and bile ducts, and produced up to 15.1% (mean, 12.0% ± 2.0%) replacement of total liver mass at 6 months after cell transplantation. These results were obtained in a normal liver background under nonselective conditions. This study is the first to show a high level of long-term liver replacement with cryopreserved fetal liver cells, an essential requirement for future clinical applications.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Approximately 30,000 people die each year of end-stage liver disease in the United States [1]. Orthotopic liver transplantation is currently the only therapeutic option for patients with end-stage chronic liver disease and for severe acute liver failure [2, 3]. However, a fundamental limitation of this therapy has been donor organ scarcity [4]. During the last decade, attention has been focused on the possibility to restore liver function through cell transplantation, especially hepatocyte transplantation. There are several advantages of hepatocyte transplantation: (a) hepatocytes from a single donor can be used for multiple recipients; (b) the procedure does not require surgery, and the cost could be much less than whole organ transplants; and (c) hepatocytes can be cryopreserved (with great difficulty, however) [5, 6]. However, a major limitation of hepatocyte transplantation is that not more than 1%–2% of liver mass can be replaced by transplanted hepatocytes [7]. In addition, after their transplantation, mature hepatocytes do not proliferate significantly [8, 9], except under very specialized conditions in which there is extensive and continuous liver injury or damage and the transplanted cells have a strong selective advantage in the recipient, or proliferation of host hepatocytes is blocked by chemical injury or x-irradiation [10–17]. Therefore, in recent years, there has been much interest in finding alternative sources of transplantable hepatic cells with the ability to proliferate and repopulate the recipient liver under more normal (nonselective) conditions.

In recent studies, we have reported that freshly isolated unfractionated embryonic day (ED) 14 fetal liver cells differentiate into both hepatocytes and bile duct epithelial cells; after transplantation, they become structurally and functionally incorporated into the hepatic parenchyma, replace 23%–24% of hepatic mass, and remain viable long-term in a normal host recipient liver [9, 18, 19]. The high level of long-term liver replacement by fetal liver stem/progenitor cells (FLSPCs) was attributable to a greater proliferative activity of transplanted cells and reduced apoptosis of their progeny compared with host hepatocytes, coupled with increased apoptosis of host hepatocytes immediately adjacent to transplanted cells [19]. This represents a process referred to as cell-cell competition [19], as reported previously in Drosophila during wing development [20, 21].

The repopulation level achieved with freshly isolated FLSPCs should be sufficient for most clinical applications. However, to reach clinical potential for FLSPCs to be transplanted, these cells need to be cryopreserved for significant periods (months to years) so that they can be banked for future use as needed. However, little is known about the properties of FLSPCs after cryopreservation and thawing. In the present study, we have cryopreserved fetal liver cells by methods similar to those used for bone marrow and cord blood cells; after thawing, these cells proliferate actively and express genes in both the hepatocytic and cholangiocytic lineages. In addition, cell transplantation experiments demonstrate differentiation of cryopreserved FLSPCs into mature hepatocytic cords and bile ducts, and they exhibit long-term liver repopulation, comparable to that obtained with freshly isolated cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Materials
Chemicals and reagents were from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com) and DAKO (Carpinteria, CA, http://www.dako.com); collagenase type 1 was from Worthington Biochemical Corporation (Lakewood, NJ, http://www.worthington-biochem.com); cell culture medium and supplements were from Gibco (Grand Island, NY, http://www.invitrogen.com); hormones were from Peprotech Inc. (Rocky Hill, NJ, http://www.peprotech.com) or Upstate Biotechnology (Lake Placid, NY, http://www.upstatebiotech.com); and fetal calf serum (FCS) was from Gemini Bio-Products (Woodland, CA, http://www.gembio.com).

Animals
Pregnant, ED14 dipeptidyl peptidase IV (DPPIV)+ Fisher (F) 344 rats, purchased from Taconic Farms (German Town, NY, http://www.taconic.com), were used to prepare fetal liver cells. DPPIV– (mutant) F344 rats were provided by the Special Animal Core of the Liver Research Center, Albert Einstein College of Medicine. All animal studies were conducted under protocols approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine in accordance with NIH guidelines.

Isolation of FLSPCs
Unfractionated fetal liver stem/progenitor cells were isolated from ED14 fetal livers of DPPIV+ pregnant F344 rats, as described previously [18]. Cell viability was >98%.

Cryopreservation and Thawing of FLSPCs
After cell isolation, FLSPC preparations were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 50% FCS and 10% dimethyl sulfoxide on ice for 20 minutes and then placed in cryogenic vials and transferred to a –80°C freezer for 2 days. Cryovials were then stored in liquid nitrogen for 1–20 months. FLSPCs were rapidly thawed in a 37°C water bath and transferred to DMEM containing 10% FCS. Cell viability was 70.1% ± 4.1%.

Cell Culture
After cryopreservation (20 months) and thawing, 0.5–1.5 x 106 viable unfractionated FLSPCs were plated on gelatin-coated two-well chamber slides or six-well plates. Plated cells were incubated in DMEM containing 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin for 24 hours, at which time the medium was switched to hormonally defined hepatocyte growth medium plus 10 ng/ml epidermal growth factor and 10 ng/ml fibroblast growth factor and changed every day thereafter [22].

Immunohistochemical Detection of {alpha}-Fetoprotein and Cytokeratin-19
Cytospins of unfractionated FLSPCs before and after cryopreservation and chamber slides of cultured FLSPCs on day 5 after cell plating were stained with sheep anti-{alpha}-fetoprotein (anti-AFP) (Nordic Immunological Laboratories, Tilburg, Netherlands, http://www.nordiclabs.nl) and mouse anti-cytokeratin-19 (anti-CK-19) (Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk). Secondary antibodies included CyTm2-conjugated donkey anti-sheep IgG and CyTm3-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com).

Immunohistochemistry for Ki-67, UDP-Glucuronosyltransferase, Albumin, and Asialoglycoprotein Receptor

Ki-67 Staining.   On days 1, 3, and 5 after cell plating, cultured FLSPCs were released from the chamber slides using trypsin/EDTA digestion and fixed to cytospin slides. Cytospins were stained with mouse anti-Ki-67 (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), followed by CyTm3-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratories).

UDP-Glucuronosyltransferase, Albumin, and Asialoglycoprotein Receptor Detection.   On day 5 after cell plating, chamber slides were stained with rabbit anti-UDP-glucuronosyltransferase (anti-UGT1A1) (kindly provided by Dr. J. Roy-Chowdhury, Liver Research Center, Albert Einstein College of Medicine), rabbit anti-albumin (ICN Biomedicals, Inc., Aurora, OH, http://www.mpbio.com) or rabbit anti-asialoglycoprotein receptor (anti-ASGPR) (kindly provided by Dr. R. Stockert, Liver Research Center, Albert Einstein College of Medicine). Secondary antibody was CyTm2-conjugated donkey anti-rabbit IgG or CyTm3-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories).

DPPIV Enzyme Histochemistry
Cytospins from cultured FLSPCs at days 1, 2, 3, 4, and 5 after cell plating were used for histochemical analysis of DPPIV enzyme expression, as previously reported [9].

Western Blot Analysis for Detection of Albumin
Cell culture medium in six-well plates was changed for the last time on day 3 after cell plating and collected 2 days later. Proteins in the culture supernatant were resolved by 10% reduced SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, using the Semi-Dry Transfer Cell system (Bio-Rad, Hercules, CA, http://www.bio-rad.com). After blocking in 5% nonfat dry milk, the membrane was incubated with rabbit anti-albumin (ICN Biochemicals). Secondary antibody was horseradish peroxidase-conjugated donkey anti-rabbit IgG (GE Healthcare-Amersham Biosciences, Little Chalfont Buckinghamshire, U.K., http://www.amersham.com). Horseradish peroxidase was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, http://www.piercenet.com).

Detection of Liver-Specific Genes Using Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted using Trizol reagent, following the manufacturer's protocol. RNA was reverse-transcribed using SuperScript III reverse transcriptase and oligo(dT) primers (supplemental online Table 1) to synthesize cDNA (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). cDNA was amplified by AmpliTaq Gold DNA polymerase (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) for 10 minutes at 95°C, followed by 20–30 cycles at 94°C for 30 seconds, 55°C for 60 seconds, 72°C for 60 seconds, and a final cycle of 72°C for 7 minutes.


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  		Table 1. Kinetics of liver repopulation by transplanted cryopreserved embryonic day 14 fetal liver cells after thawing into normal rats in conjunction with partial hepatectomy

 
Cell Transplantation
After cryopreservation (1–18 months) and thawing, different FLSPC preparations (0.5–3.0 x 107 viable cells) were pooled and infused through the portal vein into normal DPPIV– F344 rats in conjunction with two-thirds partial hepatectomy (PH).

Determination of Liver Repopulation
DPPIV expression was determined by enzyme histochemistry in liver cryosections, and liver repopulation was quantified by computerized scanning of liver sections, as previously reported [19].


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Phenotypic Characterization of Unfractionated ED14 Fetal Liver Cells Before and After Cryopreservation
In previous studies, we showed that ED14 fetal liver stem/progenitor cells contain three subpopulations: (a) cells expressing AFP, (b) cells expressing CK-19, or (c) cells expressing both AFP and CK-19 [18]. To characterize the cells to be transplanted after cryopreservation, immunohistochemical analysis for AFP and CK-19 from 14 cell preparations of freshly isolated fetal livers was performed. Unfractionated ED14 fetal liver cells contained 3.8% AFP+, 2.1% CK-19+, and 2.4% AFP+/CK-19+ cells (supplemental online Table 2).

In our previous study [18], we concluded that fetal liver epithelial cells dually marked for both the hepatocytic and bile duct epithelial lineages (i.e., AFP+/CK-19+ cells) effectively repopulate the normal adult rat liver [9, 19]. Therefore, fetal liver cell preparations showing a low percentage of AFP+/CK-19+ cells (supplemental online Table 2, preparations 4, 8, and 10) were not used for transplantation studies. The mean percentage of viable AFP+/CK19+ cells before cryopreservation was 2.4% ± 0.4%; after cryopreservation and thawing, it was maintained at 2.3% (supplemental online Table 2; supplemental online Fig. 1).


Figure 1
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  		Figure 1. Proliferation of cryopreserved embryonic day 14 fetal liver stem/progenitor cells in culture. To investigate proliferative activity (Ki-67 expression; red), cryopreserved unfractionated fetal liver cells were thawed after 20 months and cultured for up to 5 days. One day after cell plating, fetal liver cells exhibited a proliferation rate of 49.7% (A), which was reduced to 37.4% on day 3 (B) and remained unchanged at day 5 (34.7%) (C). 4,6-Diamidino-2-phenylindole was used for nuclear staining (blue). Original magnification, x600.

 
Properties of Cryopreserved ED14 Fetal Liver Cells in Culture
In the present study, we investigated the proliferative activity and differentiation potential in cell culture of fetal liver cells after long-term cryopreservation. After isolation, ED14 fetal liver cells were transferred and stored in liquid nitrogen for 20 months.

Proliferative Activity
After cryopreservation for 20 months and thawing, unfractionated fetal liver cells were cultured for 5 days. Using immunohistochemistry for Ki-67, cryopreserved cells exhibited a proliferation rate of 49.7% on day 1 after cell plating (Fig. 1A), which was maintained at 37.4% on day 3 (Fig. 1B) and was essentially unchanged on day 5 (34.7%) (Fig. 1C). This proliferative activity is similar to that obtained with freshly isolated cells in our previous studies [22].

Differentiation Capacity
To follow the differentiation of cryopreserved cells, we analyzed the expression of DPPIV. Cultured fetal liver cells were negative for DPPIV for the first 2 days in culture; however, on day 3 (Fig. 2A), a few DPPIV+ cells were detected. Both the number of DPPIV+ cells and the intensity of DPPIV expression increased sequentially on day 4 (Fig. 2B) and day 5 (Fig. 2C).


Figure 2
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  		Figure 2. Differentiation of cryopreserved fetal liver cells in culture. To follow the differentiation of cells cryopreserved for 20 months and then placed in culture, expression of dipeptidyl peptidase IV (DPPIV), which is not expressed in early fetal liver development, was analyzed using enzyme histochemistry. Cultured fetal liver cells were negative for DPPIV during the first 2 days; however, on day 3 (A), a few DPPIV+ cells were detected (arrow). Both the number of DPPIV+ cells and intensity of DPPIV expression increased on day 4 (B) and day 5 (C). Original magnification, x600.

 
Phenotypic characterization using immunohistochemistry for AFP and CK-19 showed that the majority of cultured cells were positive for AFP and CK-19 on day 5 (Fig. 3). In addition, more than 80% of the cells expressed the UDP-glucuronosyltransferase (Fig. 4A, 4D), albumin (Fig. 4B, 4E), and the asialoglycoprotein receptor (Fig. 4C, 4F) (all unique hepatocyte-specific functions).


Figure 3
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  		Figure 3. Bipotent phenotype of cryopreserved embryonic day 14 fetal liver cells in vitro. Using immunohistochemistry for AFP and CK-19, the majority of cultured cells (after cryopreservation for 20 months) were positive for both AFP (A, D) and CK-19 (B, E) at day 5. Merged images are shown in (C) and (F). Nuclei were identified by 4,6-diamidino-2-phenylindole. Original magnification, x900. Abbreviations: AFP, {alpha}-fetoprotein; CK, cytokeratin.

 


Figure 4
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  		Figure 4. Hepatocytic marker expression and albumin secretion by 20-month cryopreserved embryonic day (ED) 14 fetal liver cells in culture. (A–F): More than 80% of the cells expressed unique hepatocyte-specific functions in cell culture at day 5: UGT1A1 (A, D), albumin (B, E), and ASGPR (C, F). 4,6-Diamidino-2-phenylindole was used for nuclear staining (blue). (A–C): Original magnification, x600. (D–F): Original magnification, x900. (G): Albumin production by cryopreserved fetal liver cells in culture. Twenty months after cryopreservation, 1.5 x 106 viable unfractionated ED14 fetal liver cells were cultured in hormonally defined hepatocyte growth medium. Culture medium was changed for the last time on day 3 after cell plating, and supernatant was collected for Western blot analysis 2 days later. Control medium is shown in the left lane, and the lanes labeled 1–6 are from six different cell cultures. Abbreviations: ASGPR, asialoglycoprotein receptor; Cont., control; UGT1A1, UDP-glucuronosyltransferase.

 
Messenger RNA from ED14 fetal liver cells after thawing was compared with mRNA of cryopreserved cells in culture for 5 days and from adult liver (Fig. 5). AFP mRNA was highly expressed in all samples. Albumin, CK-19, hematopoietically expressed homeobox (HEX), and hepatocyte nuclear factor (HNF)3ß mRNAs were weakly expressed at ED14 (cryopreserved cells after thawing) but strongly expressed after cell culture and in adult liver. Glucose-6-phosphatase (G6Pase), carbamyl phosphate synthetase (CPS-1), HNF4{alpha}, tyrosine aminotransferase (TAT), and oncostatin M receptor (OSMR) mRNAs were also negative in ED14 fetal liver cells after cryopreservation but were expressed on day 5 in cell culture and in adult liver tissue. Tryptophan oxygenase (TRPO) was expressed only in adult liver. Cryopreserved fetal liver cells (before and after cell culture) were negative for cytochrome (CYP) 2E1 and CYP7A1 but positive for CYP3A1 at 5 days in culture in the presence of dexamethasone (Decadron; Merck & Co., Inc., Whitehouse Station, NJ, http://www.merck.com). Adult hepatocytes exhibited all of these differentiated functions.


Figure 5
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  		Figure 5. Gene expression profile of embryonic day (ED) 14 fetal liver cells after cryopreservation. Using reverse transcription-polymerase chain reaction analysis, mRNA from ED14 fetal liver cells after cryopreservation for 20 months (lane 1) was compared with mRNA after cell culture on day 5 (lanes 2 and 3) and from adult liver (lane 4), using GAPDH as internal control. Abbreviations: AFP, {alpha}-fetoprotein; Alb, albumin; CK, cytokeratin; CPS-1, carbamyl phosphate synthetase; CYP, cytochrome; G6Pase, glucose-6-phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEX, hematopoietically expressed homeobox; HNF, hepatocyte nuclear factor; OSMR, oncostatin M receptor; TAT, tyrosine aminotransferase; TRPO, tryptophan oxygenase.

 
Albumin Secretion
An important property of fetal liver stem/progenitor cells is that they differentiate into hepatocytes with biologic function. After cryopreservation and culture, supernatants from several ED14 fetal liver cell cultures were collected at days 3–5, and albumin secretion into the medium was detected by Western blot analysis. All culture supernatants contained considerable quantities of albumin (Fig. 4G).

In summary, cryopreserved ED14 fetal liver cells exhibited a phenotype similar to that of freshly isolated ED14 FLSPCs, that is, they proliferated rapidly in culture and differentiated along the hepatocytic lineage, as demonstrated by showing a large variety of hepatocyte-specific markers and functions.

Repopulation of the Normal Liver by Cryopreserved ED14 Fetal Liver Stem/Progenitor Cells
In a pilot experiment, we transplanted 0.5 x 107 viable cells isolated from DPPIV+ ED14 rat fetal livers and cryopreserved for 1 month into the liver of syngeneic DPPIV– rats under nonselective conditions (Table 1, left column). Transplanted DPPIV+ cells differentiated into hepatocytes and cholangiocytes and became incorporated into the parenchymal plates and bile ducts. In addition, the number and size of DPPIV+ cell clusters increased progressively for up to 6 months after cell transplantation (Fig. 6A–6C).


Figure 6
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  		Figure 6. Liver repopulation in normal rats by transplanted embryonic day (ED) 14 fetal liver stem/progenitor cells after cryopreservation. (A–C): After cryopreservation for 1 month and thawing, 0.5 x 107 viable ED14 fetal liver cells from dipeptidyl peptidase IV (DPPIV)+ F344 rats were transplanted into the portal vein of DPPIV– F344 rats in conjunction with two-thirds partial hepatectomy. Animals were sacrificed after 2 (A), 4 (B), and 6 months (C), and the presence of transplanted cells was detected by DPPIV enzyme histochemistry. Original magnification, x200. (D): Whole liver section from one animal transplanted with 2.5 x 107 ED14 fetal liver cells and sacrificed at 6 months after transplantation of pooled cells cryopreserved for 1–18 months. Comparable results were found in the other two animals (Table 1).

 
To precisely quantitate liver replacement over time, different cell preparations from freshly isolated ED14 fetal liver cells were cryopreserved for 1–18 months and pooled after rapid thawing before transplantation. In several experiments, listed in the center column of Table 1, 1.1–1.5 x 107 viable cells were transplanted into the normal adult rat liver in conjunction with PH, and the percentage of liver replacement was determined 2, 4, and 6 months after cell transplantation. As shown in Table 1, we observed substantial proliferation of transplanted cells within 2 months, as evidenced by numerous clusters containing up to 100 cells, increasing to 100–500 cells per cluster at 4 months and >500 cells per cluster at 6 months. The number and size of clusters increased continuously over time, and the percentage repopulation also increased throughout time, with up to 15.1% (mean, 12.0% ± 2.0%) liver replacement with 2.5–3.0 x 107 transplanted ED14 fetal liver cells at 6 months (determined by scanning two sections from two different areas/lobes from three different cell transplanted rats) (Fig. 6D; Table 1, right column). This level of long-term repopulation is similar to that obtained with freshly isolated cells in previous studies when using the same number of transplanted freshly isolated ED14 fetal liver cells [19] (Fig. 2A).

In two experiments, pooled preparations of unfractionated fetal liver cells, stored in liquid nitrogen for 4–7 months versus 12–15 months and 1–2 months versus 12–15 months, respectively, were used for cell transplantations (Table 1, center column). No significant difference was observed in liver repopulation by these various pooled cell preparations at either 4 months after cell transplantation (first experiment) or 6 months after cell transplantation (second experiment). These results showed that cryopreserved fetal liver cells can be stored for a long time without significant loss of their in vivo repopulation capacity.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
Stem/progenitor cells are a promising source for liver reconstitution by cell transplantation. Various reports have demonstrated that hematopoietic stem cells derived from the bone marrow (BM) engraft in the liver and differentiate into hepatocytes, but in the majority of studies, the yield of hematopoietic cell-derived hepatocytes was less than 1% of the recipient hepatocytes [23]. Other studies have implicated mesenchymal stem cells or nonhematopoietic multipotent adult progenitor cells as a source of cells in the BM capable of differentiating into hepatocytes after engraftment into the liver [24].

Currently, fetal liver stem/progenitor cells are the only known cell type that can effectively repopulate the normal liver for the long-term under nonselective conditions and can restore normal liver tissue mass and function at a level that should be sufficient to treat a wide variety of metabolic and other liver disorders [9, 19]. However, for general therapeutic use, isolated stem/progenitor cells will need to be cryopreserved for an extended period (months to years) so that they can be appropriately distributed on a prioritized basis. In addition, cellular cryopreservation would allow for extensive testing and validation of cell sources to assess the safety and efficacy of the cells.

In the present study, we used a standard method for freezing and thawing of the cells. The viability of fetal liver cells after cryopreservation and thawing was 70.1%. A number of studies have reported different methods and strategies for effective cryopreservation of adult hepatocytes resulting in high cell viability (up to 90%) immediately after thawing [25–29]; however, the viability of the cells decreased rapidly during cryopreservation, as demonstrated by loss of attachment rate and hepatic functions [27, 30]. In contrast, Ikeda et al. showed that a subpopulation of small hepatocytes maintain their growth ability and differentiated hepatic functions in cell culture, even after long-term cryopreservation [31]. Using the hypothermic preservation solution HypoThermosol, another study demonstrated that isolated hepatocytes showed high viability, showed long-term hepatospecific function (albumin secretion and urea synthesis), and responded to cytokine challenge (cellular response to interleukin-6) after cryopreservation [32]. Thus, several parameters are critical for effective cryopreservation/thawing of cells: the choice of freezing medium, addition of supplements and cryoprotectants (and their concentrations), cooling and thawing rates, and slow addition/removal of freezing solution [33]. However, with fetal liver cells, much simpler cryopreservation methods can be used [34, 35 and the present study], comparable to those used for human bone marrow or cord blood hematopoietic stem cells [36].

To characterize the quality of the isolated cells after cryopreservation, cell culture studies were performed to determine the proliferative activity and differentiation potential of the cells. Freshly isolated ED14 fetal liver cells, cryopreserved for 20 months, were able to proliferate massively in culture, as assessed by immunohistochemistry for Ki-67, ranging from 49.7% on day 1 to 34.7% on day 5, levels similar to those obtained with freshly isolated cells (46.8% on day 1 to 20.5% on day 4), as assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation [22] (our unpublished data). Similar results were obtained with early human fetal liver cells in culture [35].

The ability to differentiate into mature hepatocytes is a critical property of fetal liver stem/progenitor cells, as hepatospecific functions are essential for use in clinical applications. Therefore, we performed extensive studies in vitro to show the differentiation potential and function of ED14 fetal liver cells after long-term cryopreservation. DPPIV, a type II membrane glycoprotein, is expressed during late fetal development (starting at ED17) [37] and is localized mainly to the bile canalicular membrane in adult liver parenchyma [38–40]. In the present study, DPPIV was first detected in cryopreserved fetal liver cells on day 3 in culture and increased thereafter, indicating differentiation of the cells.

Immunohistochemical analysis (confirmed by reverse transcription-polymerase chain reaction [RT-PCR]) showed that the majority of the cultured cells were positive for AFP, albumin, and CK-19 at day 5, showing potential bipotency or dual lineage capacity for these cells. However, more than 80% of the fetal liver cells in culture expressed UDP-glucuronosyltransferase, an enzyme that specifically conjugates bilirubin into mono- and diglucuronides in hepatocytes [41], and another unique hepatocyte-specific marker, the asialoglycoprotein receptor [42], which is first expressed in rat fetal liver at ED17 [37]. In addition, using RT-PCR analysis, G6Pase, CPS-1, HNF4{alpha}, TAT, and OSMR mRNAs were not detected in ED14 fetal liver cells after cryopreservation; however, they were all strongly expressed at day 5 in cell culture, indicating hepatocytic lineage maturation of fetal liver cells. Finally, physiologic function of cryopreserved fetal liver cells in culture was demonstrated by secretion of albumin, the most abundant protein synthesized by hepatocytes. Taken together, ED14 fetal liver cells retained their differentiation potential after long-term cryopreservation. However, the differentiation process into mature hepatocytes was not completed at day 5 in cell culture (TRPO, CYP2E1, and CYP7A1 mRNAs were not expressed).

Most importantly, we also showed that cryopreserved fetal liver stem/progenitor cells retained their proliferative activity and differentiation potential after cell transplantation. Cryopreserved fetal liver cells engrafted and proliferated continuously for up to 6 months and differentiated into both hepatocytes and bile duct cells. The length of cryopreservation time did not significantly influence the repopulation capacity. The level of repopulation reached a mean of 12.0% of the normal liver mass after transplantation of 2.5–3.0 x 107 cells, a level comparable to 23.5% liver replacement with 4–6 x 107 freshly isolated cells [19]. These data showed that the bipotential stem/progenitor cell population (AFP+/CK-19+), which is responsible for liver replacement, retained proliferative and differentiation properties after cryopreservation. This correlates with the immunohistochemical analysis for AFP and CK-19, showing that the percentage of AFP+/CK-19+ cells was maintained after cryopreservation/thawing similar to that before cryopreservation (2.3% vs. 2.4%).

Fetal liver stem/progenitor cells have a higher proliferative activity than adult hepatocytes both in vitro and after their transplantation. Despite their higher proliferative activity, we have not found malignant transformation of fetal liver cells for up to 18 months after their transplantation into the normal adult rat liver (our unpublished data). We have also studied transplantation of fetal liver stem/progenitor cells in the retrorsine-treated rat liver, where transplanted cell proliferation and liver repopulation are markedly enhanced. In the retrorsine/PH model, we have obtained >95% repopulation of the adult liver at 1 year after transplanting cryopreserved ED14 fetal liver stem/progenitor cells (supplemental online Fig. 2), yet we have still not seen malignant transformation of the liver, even under these circumstances of prolonged liver injury in which host hepatocytes are incapable of dividing.

Our study demonstrates the highest level of long-term liver replacement achieved with cryopreserved fetal liver stem/progenitor cells conducted to date, and these studies were performed under nonselective conditions. Malhi et al. showed that human fetal liver cells can engraft and survive after transplantation into SCID mouse livers; however, liver repopulation was very low [43]. In another report, cryopreserved early human fetal liver cells engrafted and proliferated in athymic mice for up to 6 weeks, after which they became quiescent [35]. The percentage of repopulation was significantly higher than that observed by Malhi et al. [43], and, interestingly, one animal exhibited up to 10% repopulation, although in the majority of animals, repopulation was in a range between 0.05% and 1.0% [35].

In summary, ED14 fetal liver stem/progenitor cells can be cryopreserved for a long time without loss of their high proliferative activity and differentiation potential in cell culture. After thawing, rat fetal liver cells are able to proliferate for an extended period after cell transplantation and differentiate into hepatocytes and bile duct cells. The level of long-term liver replacement achieved with cryopreserved fetal liver stem/progenitor cells is comparable to that obtained with freshly isolated cells and should be sufficient to treat most inherited metabolic disorders of the liver.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We thank Anna Caponigro for secretarial assistance. M.O. and A.M. contributed equally to this work. This work was supported in part by NIH Grants R01-DK17609 and P30-DK41296 (D.A.S.).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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