Transfer and Stable Transgene Expression of a Mammalian Artificial Chromosome into Bone Marrow-Derived Human Mesenchymal Stem Cells
http://www.100md.com
《干细胞学杂志》
Chromos Molecular Systems Inc., Burnaby, British Columbia, Canada
Key Words. Mammalian artificial chromosomes ? ACEs ? Gene therapy ? Cell therapy ? Mesenchymal stem cells
Sandra Vanderbyl, MSc, Chromos Molecular Systems Inc., 8081 Lougheed Highway, Burnaby, British Columbia, Canada V5A 1W9. Telephone: 604-415-7100; Fax: 604-415-7151; e-mail: svanderbyl@chromos.com
ABSTRACT
Recent events in gene therapy applications have driven the development of vectors that maintain stable expression of therapeutic genes without the risk of cell transformation or the stimulation of the host immune system . However despite advances, current eukaryotic viral vector systems remain problematic. For example, integration into the host chromosome by retroviral-based vectors may lead to variegated gene expression, insertional mutagenesis, and oncogenesis . Similar concerns have also been raised regarding the integration of adeno-associated virus vectors and their association with chromosomal deletions and other rearrangements that are frequently located on human chromosome 19 . Furthermore, it has been demonstrated that retroviruses preferentially integrate near transcription start regions thus raising the likelihood of insertional mutagenesis with retrovirus-based vectors . Transient expression of therapeutic products is also a concern. Most notably, using nonintegrating vector systems such as adenoviral-based vectors may elicit a host immunological response to the vector, including death .
Artificial chromosomes (ACEs) are being investigated as an alternative to current eukaryotic viral vector systems. Hadlaczky and colleagues developed a unique methodology to construct mammalian ACEs through the induction of large scale amplifications of "satellite" DNA sequences in pericentromeric heterochromatin . De novo centromeres and dicentric chromosomes were formed upon the integration of exogenous DNA sequences into the specific regions of acrocentric chromosomes—those containing both pericentromeric heterochromatin and the tandemly repeated ribosomal genes (rDNA). Ensuing breakage during mitosis generated new chromosomes ranging in size from 10 to 360 megabases . Considering that these ACEs, termed satellite DNA-based artificial chromosomes (SATACs), can be manipulated or engineered to contain a variety of exogenous sequences, they present a unique opportunity for a variety of applications, including gene therapy . SATACs have recently been engineered to contain multiple recombination acceptor sites in order to facilitate the insertion of desired transgenes onto the ACEs. These engineered SATACs are commonly referred to as Platform ACEs or ACEs.
ACEs are safe, stable systems, and many numerous reviews have addressed their utility. In addition to ease of loading genes onto Platform ACEs, they can be isolated and stably transferred. This further expands their utility. As part of the overall ACE System, multiple transgenes (cDNAs or larger genomic encoding regions) can be efficiently targeted onto the Platform ACE through site-specific DNA recombination . Platform ACEs and transgene-loaded ACEs can be isolated by flow cytometry to high purities (>95%) and yields (>1,000,000 ACEs/hr) and can be transferred in vitro into a variety of mammalian cell lines and primary cells by cationic lipids and dendrimers . ACEs can also be used to generate transgenic mice through microinjection of purified ACEs into fertilized oocytes . Stability is exemplified by the fact the resulting transgenic mice were shown to stably maintain the ACE through four generations of the germline .
Human mesenchymal stem cells (hMSCs) have emerged as a promising strategy for ex vivo gene therapy . Improvements in isolation, in vitro cultivation of hMSCs , and in vivo studies on differentiation into functional cells of multiple tissue types make them attractive delivery vehicles for autologous cell-mediated gene therapy. Characteristics that make them appealing cell hosts include ease of purification from clinical bone marrow (BM) aspirates (frequency of 1/105) and the ability to culture and expand hMSCs in vitro without apparent loss of differentiation potential into multiple cell types . Additionally MSCs have been shown to confer immune privilege by not presenting alloantigens and by suppressing T-cell lymphocyte proliferation . These features may enable a cellular therapy scenario whereby hMSCs can be derived from a "universal" donor (irrespective of their major histocompatability complex haplotype), expanded, and prepared as an "off-the-shelf" reagent.
hMSCs are being evaluated as cellular delivery vehicles for therapeutic genes for a variety of clinical indications. This list includes the replacement of genes to correct acquired or inherited disorders of bone, muscle, or cartilage by transfer of genes encoding bone morphogenic proteins -2, -4, -9 , the transfer of genes encoding the blood coagulation factors VIII and IX for the treatment of hemophilia , human growth hormone , insulin-like growth factor 1 , and interleukin-3 . This list also includes: erythropoietin , -l-iduronidase for treatment of mucopolysaccharidosis type I , pro2 collagen (I) for treatment of osteogenesis imperfecta , sox9 to enhance chondrogenesis, and interferon to treat tumors . In several of these studies retroviral-based vectors were utilized, thereby increasing the risk of inducing insertional mutagenesis. The risk of insertional mutagenesis can be reduced prior to patient treatment by physical screening of selected recombinants for vector integration into genetically safe regions; however, this approach is problematic when transducing committed stem cells (SCs) with limited cell doublings. Furthermore, the integrated vectors often result in pronounced transgene silencing leading to short-term therapeutic gain. Theoretically, the ACE System would overcome these issues as ACEs are nonintegrating and can be engineered to optimize long-term transgene expression.
Cell therapy regimes are restricted by the ability to target host cells, the expression and stability of the inserted constructs, the delivery efficiency of vectors, and safety. When considering the application of ACE-based cell therapies in the clinic, a limitation that requires further investigation is the delivery efficiency of protein-DNA complexes. When compared to viral vectors, which can reach transduction efficiencies of 100%, the transfer efficiency of the ACEs is at least an order of magnitude lower . Therefore, strategies that facilitate the enrichment of cells harboring a transferred ACE must be simultaneously developed. To this end, the use of human cell surface or drug selectable marker genes is being investigated as a means to enrich for the ACE-containing cells within a population.
In addition to the enrichment and ex vivo delivery strategies for ACE-containing cells, we are currently initiating animal studies to investigate the potential for application of ACE-based cell therapies in disease models along with the relative safety and tolerability of these modified cells. With respect to the latter, current data suggest that ACEs are mitotically and meiotically stable, nontumorigenic, and nonmutagenic, as transgenic mice containing an ACE were phenotypically normal, as were their offspring through four generations . Although these observations will need to be substantiated in the context of cellular therapies, our current plans include these and additional cellular assays to determine cell fate, stability of the ACE, duration of transgene expression, and immune response. Current animal studies focus on the utility of ACE-modified MSCs in a small animal model of disease that, if successful, will represent the first demonstration of a therapeutic benefit using a chromosome-based ex vivo cell therapy.
In this work we evaluated the ability of BM-derived hMSCs to be genetically modified with ACEs expressing the red fluorescent protein (RFP). An intact ACE chromosome was introduced into the hMSCs, and reporter gene expression was detected in the transfected hMSCs. hMSCs containing the ACEs maintained their multipotential capacity as evidenced by their ability to be induced to differentiate along osteogenic and adipogenic lineages. Furthermore, RFP expression was detected in the differentiated cells suggesting that the ACE chromosomal environment is well suited for transgene expression in differentiating SC populations. To our knowledge this is the first report of the stable expression of a transgene introduced into human adult SCs by mammalian ACE. This finding demonstrates the potential utility of the ACE technology for human adult SC ex vivo gene therapy and provides a novel approach for a broad range of tissue engineering applications.
MATERIALS AND METHODS
ACE-Transgene Expression in hMSCs
As a first step pursuant to the use of ACEs in a gene therapy context, we tested the ability to introduce flow-sorted and concentrated RFP-ACEs into a purified hMSC population. The RFP-ACEs were transfected into limited passaged hMSCs using commercially available transfection reagents. After transfer of the RFP-ACEs, the cells were passaged in 10-cm dishes, and the level of transgene expression (RFP fluorescence) was monitored by flow cytometry. From 4 to 5 days post-transfection, RFP expression was detected in 11 ± 4% (n = 3; scoring a minimum of 10,000 cells) of the total hMSC population. Intact RFP-ACEs, as determined by FISH analysis, were detected in 5 ± 3% (n = 2; scoring a minimum of 50 metaphase chromosome spreads) of total hMSC population. A representative example of FISH analysis of the RFP-ACEs-modified hMSCs can be seen in Figure 1A. Since the frequency of intact delivered RFP-ACEs/transfected hMSC, as determined by FISH analyses, was not statistically different from the observed frequency of RFP-expressing hMSCs in the total cell population that was transfected (p-value 0.05), this suggested that in general the transfection of the RFP-ACEs into the hMSCs resulted in the delivery of an intact chromosome.
Figure 1. FISH analyses. A) Untransfected control hMSCs (i) and ACE-modified hMSCs (ii) were incubated with colchicine, fixed to microscope slides, and hybridized to digoxigenin-labeled mouse major satellite DNA (red signal). Note that the ACE is intact and is maintained as an autonomous chromosome. B) ACE-modified hMSCs were induced to differentiate into either osteocytes (i) or adipocytes (ii) analysis done with interphase cells. Cells were fixed and hybridized to digoxigenin-labeled mouse major satellite DNA (red signal).
ACE-Transgene Expression During hMSC Differentiation
We evaluated whether the hMSCs carrying the RFP-ACEs still maintained their potential to differentiate into unique cell lineages. To do so, we elected to monitor reporter gene expression in the hMSC population as the cells differentiated along adipogenic and osteogenic lineages. These two pathways were chosen because the differentiated cells were visually distinctive, based on microscopic examination, and simple biochemical identification assays were available—staining lipids with Oil Red "O" or measuring insoluble calcium deposition to identify adipocytes or osteocytes, respectively. Initially differentiation was monitored in heterogeneous cell populations prior to enrichment of RFP-ACEs containing hMSCs (population with 11% RFP-expressing hMSCs). In this case, the RFP-ACE-transfected hMSCs appear to differentiate at a rate comparable to the untransfected controls. Greater than 95% of the cells had differentiated 2–3 weeks post-induction into either adipogenic or osteogenic lineages (visual inspection, data not shown). Furthermore, during differentiation RFP expression was maintained (visual inspection). FISH analyses were performed on terminally differentiated cultures 2–3 weeks post-induction in order to evaluate the integrity of the transferred RFP-ACEs during differentiation (Fig. 1B). The frequencies of RFP-ACEs were scored as 25% ± 7% (n = 2) in adipocytes and 6% ± 4% (n = 2) in osteocytes. These preliminary experiments indicate that the hMSCs stably maintained RFP-ACEs and concomitant RFP expression after osteogenic and adipogenic differentiation (Fig. 2A, 2B).
Figure 2. A) RFP-expressing adipocytes at 2 weeks post induction in the adipocyte differentiation pathway. Panel (i): Phase microscopic view from a representative field. Panel (ii): RFP-expressing cells from the identical microscopic field as depicted in panel (i). B) RFP-expressing osteocytes at 2 weeks post induction in the osteocyte differentiation pathway. Panel (i): Phase microscopic view from a representative field. Panel (ii): RFP-expressing cells from the identical microscopic field as depicted in panel (i).
RT-PCR analyses of selected gene markers (PPAR2, LDL, osteopontin) from induced and non-induced cultures were performed to further confirm the differentiated phenotype of the mesenchymal cultures containing the RFP-ACEs. Previous reports have utilized the expression of PPAR2 and LDL as hallmarks of adipogenic differentiation and osteopontin as a biomarker for osteogenic differentiation .
In order to detect transgene expression and monitor cytogenetic events during differentiation, the RFP-expressing hMSCs were enriched by flow cytometry 3–5 days post-transfection, and plated into three sets of wells. One sample (non-induced control) was maintained under hMSC culturing conditions, whereas parallel cultures were induced to differentiate into osteogenic and adipogenic lineages. Samples of induced cells were harvested at various times during the 3-week induction period. The cultures were analyzed by RT-PCR to measure the induced expression of lineage specific marker genes and by FISH. RT-PCRs for ?-actin transcripts were conducted as measures of RNA integrity for all culturing conditions since ?-actin expression is ubiquitous for both undifferentiated and differentiated hMSCs (top panels Fig. 3, lanes C-F). Only hMSCs induced to the adipogenic lineage expressed LDL or PPAR2 mRNA (Summary RT-PCR results , representative LDL profile Fig. 3 bottom panels I-M). RT-PCR detected expression of the LDL and PPAR2 genes in the genetically modified adipocyte cultures as early as 1 week post induction. Parallel osteogenic-induced cultures were tested for the presence of adipogenic lineage marker genes LDL or PPAR2. For all of the osteogenic-induced cultures, expression of LDL or PPAR2 genes was not detected (data not shown). Similar results showing lineage specificity were found when assessing osteogenic-induced cultures for expression of the osteogenic lineage marker, osteopontin, 13 days post-induction (Table 1). Overall, the RFP-ACE-modified hMSCs continued to express the RFP transgene and to maintain the integrity of the RFP-ACE after in vitro differentiation.
Figure 3: RT-PCR analyses using the ubiquitous cell lineage marker ?-actin (row B-F) and the specific adipose marker LPL (row I-M) from adipocyte mRNA. A ?-actin PCR product was found in all samples except for the negative PCR control. Top row: 515-bp ?-actin products. A) 100-bp ladder; B) negative control (no RNA); C) transfected hMSC (undifferentiated); D) transfected, 7-days post adipocyte induction; E) transfected, 13-days post adipocyte induction; F) nontransfected, 13-days post adipocyte induction; G) 100-bp ladder. Bottom row: 276-bp LPL products; H) 100-bp ladder; I) negative control (no RNA); J) transfected hMSC (undifferentiated); K) transfected, 7-days post adipocyte induction; L) transfected, 13-days post adipocyte induction; M) nontransfected, 13-days post adipocyte induction; N) 100-bp ladder. For summary of RT-PCR analyses refer to Table 1.
Table 1. Summary of RT-PCR of hMSCs cells
DISCUSSION
Chromos Molecular Systems, Inc. was responsible for all material and financial support for this research. We would like to thank Diane Monteith and Tom Stodola, for purifying ACEs and technical support; Harry Ledebur and Joseph Zendegui for critical discussions; and Shamila Gorjian for manuscript preparation.
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Key Words. Mammalian artificial chromosomes ? ACEs ? Gene therapy ? Cell therapy ? Mesenchymal stem cells
Sandra Vanderbyl, MSc, Chromos Molecular Systems Inc., 8081 Lougheed Highway, Burnaby, British Columbia, Canada V5A 1W9. Telephone: 604-415-7100; Fax: 604-415-7151; e-mail: svanderbyl@chromos.com
ABSTRACT
Recent events in gene therapy applications have driven the development of vectors that maintain stable expression of therapeutic genes without the risk of cell transformation or the stimulation of the host immune system . However despite advances, current eukaryotic viral vector systems remain problematic. For example, integration into the host chromosome by retroviral-based vectors may lead to variegated gene expression, insertional mutagenesis, and oncogenesis . Similar concerns have also been raised regarding the integration of adeno-associated virus vectors and their association with chromosomal deletions and other rearrangements that are frequently located on human chromosome 19 . Furthermore, it has been demonstrated that retroviruses preferentially integrate near transcription start regions thus raising the likelihood of insertional mutagenesis with retrovirus-based vectors . Transient expression of therapeutic products is also a concern. Most notably, using nonintegrating vector systems such as adenoviral-based vectors may elicit a host immunological response to the vector, including death .
Artificial chromosomes (ACEs) are being investigated as an alternative to current eukaryotic viral vector systems. Hadlaczky and colleagues developed a unique methodology to construct mammalian ACEs through the induction of large scale amplifications of "satellite" DNA sequences in pericentromeric heterochromatin . De novo centromeres and dicentric chromosomes were formed upon the integration of exogenous DNA sequences into the specific regions of acrocentric chromosomes—those containing both pericentromeric heterochromatin and the tandemly repeated ribosomal genes (rDNA). Ensuing breakage during mitosis generated new chromosomes ranging in size from 10 to 360 megabases . Considering that these ACEs, termed satellite DNA-based artificial chromosomes (SATACs), can be manipulated or engineered to contain a variety of exogenous sequences, they present a unique opportunity for a variety of applications, including gene therapy . SATACs have recently been engineered to contain multiple recombination acceptor sites in order to facilitate the insertion of desired transgenes onto the ACEs. These engineered SATACs are commonly referred to as Platform ACEs or ACEs.
ACEs are safe, stable systems, and many numerous reviews have addressed their utility. In addition to ease of loading genes onto Platform ACEs, they can be isolated and stably transferred. This further expands their utility. As part of the overall ACE System, multiple transgenes (cDNAs or larger genomic encoding regions) can be efficiently targeted onto the Platform ACE through site-specific DNA recombination . Platform ACEs and transgene-loaded ACEs can be isolated by flow cytometry to high purities (>95%) and yields (>1,000,000 ACEs/hr) and can be transferred in vitro into a variety of mammalian cell lines and primary cells by cationic lipids and dendrimers . ACEs can also be used to generate transgenic mice through microinjection of purified ACEs into fertilized oocytes . Stability is exemplified by the fact the resulting transgenic mice were shown to stably maintain the ACE through four generations of the germline .
Human mesenchymal stem cells (hMSCs) have emerged as a promising strategy for ex vivo gene therapy . Improvements in isolation, in vitro cultivation of hMSCs , and in vivo studies on differentiation into functional cells of multiple tissue types make them attractive delivery vehicles for autologous cell-mediated gene therapy. Characteristics that make them appealing cell hosts include ease of purification from clinical bone marrow (BM) aspirates (frequency of 1/105) and the ability to culture and expand hMSCs in vitro without apparent loss of differentiation potential into multiple cell types . Additionally MSCs have been shown to confer immune privilege by not presenting alloantigens and by suppressing T-cell lymphocyte proliferation . These features may enable a cellular therapy scenario whereby hMSCs can be derived from a "universal" donor (irrespective of their major histocompatability complex haplotype), expanded, and prepared as an "off-the-shelf" reagent.
hMSCs are being evaluated as cellular delivery vehicles for therapeutic genes for a variety of clinical indications. This list includes the replacement of genes to correct acquired or inherited disorders of bone, muscle, or cartilage by transfer of genes encoding bone morphogenic proteins -2, -4, -9 , the transfer of genes encoding the blood coagulation factors VIII and IX for the treatment of hemophilia , human growth hormone , insulin-like growth factor 1 , and interleukin-3 . This list also includes: erythropoietin , -l-iduronidase for treatment of mucopolysaccharidosis type I , pro2 collagen (I) for treatment of osteogenesis imperfecta , sox9 to enhance chondrogenesis, and interferon to treat tumors . In several of these studies retroviral-based vectors were utilized, thereby increasing the risk of inducing insertional mutagenesis. The risk of insertional mutagenesis can be reduced prior to patient treatment by physical screening of selected recombinants for vector integration into genetically safe regions; however, this approach is problematic when transducing committed stem cells (SCs) with limited cell doublings. Furthermore, the integrated vectors often result in pronounced transgene silencing leading to short-term therapeutic gain. Theoretically, the ACE System would overcome these issues as ACEs are nonintegrating and can be engineered to optimize long-term transgene expression.
Cell therapy regimes are restricted by the ability to target host cells, the expression and stability of the inserted constructs, the delivery efficiency of vectors, and safety. When considering the application of ACE-based cell therapies in the clinic, a limitation that requires further investigation is the delivery efficiency of protein-DNA complexes. When compared to viral vectors, which can reach transduction efficiencies of 100%, the transfer efficiency of the ACEs is at least an order of magnitude lower . Therefore, strategies that facilitate the enrichment of cells harboring a transferred ACE must be simultaneously developed. To this end, the use of human cell surface or drug selectable marker genes is being investigated as a means to enrich for the ACE-containing cells within a population.
In addition to the enrichment and ex vivo delivery strategies for ACE-containing cells, we are currently initiating animal studies to investigate the potential for application of ACE-based cell therapies in disease models along with the relative safety and tolerability of these modified cells. With respect to the latter, current data suggest that ACEs are mitotically and meiotically stable, nontumorigenic, and nonmutagenic, as transgenic mice containing an ACE were phenotypically normal, as were their offspring through four generations . Although these observations will need to be substantiated in the context of cellular therapies, our current plans include these and additional cellular assays to determine cell fate, stability of the ACE, duration of transgene expression, and immune response. Current animal studies focus on the utility of ACE-modified MSCs in a small animal model of disease that, if successful, will represent the first demonstration of a therapeutic benefit using a chromosome-based ex vivo cell therapy.
In this work we evaluated the ability of BM-derived hMSCs to be genetically modified with ACEs expressing the red fluorescent protein (RFP). An intact ACE chromosome was introduced into the hMSCs, and reporter gene expression was detected in the transfected hMSCs. hMSCs containing the ACEs maintained their multipotential capacity as evidenced by their ability to be induced to differentiate along osteogenic and adipogenic lineages. Furthermore, RFP expression was detected in the differentiated cells suggesting that the ACE chromosomal environment is well suited for transgene expression in differentiating SC populations. To our knowledge this is the first report of the stable expression of a transgene introduced into human adult SCs by mammalian ACE. This finding demonstrates the potential utility of the ACE technology for human adult SC ex vivo gene therapy and provides a novel approach for a broad range of tissue engineering applications.
MATERIALS AND METHODS
ACE-Transgene Expression in hMSCs
As a first step pursuant to the use of ACEs in a gene therapy context, we tested the ability to introduce flow-sorted and concentrated RFP-ACEs into a purified hMSC population. The RFP-ACEs were transfected into limited passaged hMSCs using commercially available transfection reagents. After transfer of the RFP-ACEs, the cells were passaged in 10-cm dishes, and the level of transgene expression (RFP fluorescence) was monitored by flow cytometry. From 4 to 5 days post-transfection, RFP expression was detected in 11 ± 4% (n = 3; scoring a minimum of 10,000 cells) of the total hMSC population. Intact RFP-ACEs, as determined by FISH analysis, were detected in 5 ± 3% (n = 2; scoring a minimum of 50 metaphase chromosome spreads) of total hMSC population. A representative example of FISH analysis of the RFP-ACEs-modified hMSCs can be seen in Figure 1A. Since the frequency of intact delivered RFP-ACEs/transfected hMSC, as determined by FISH analyses, was not statistically different from the observed frequency of RFP-expressing hMSCs in the total cell population that was transfected (p-value 0.05), this suggested that in general the transfection of the RFP-ACEs into the hMSCs resulted in the delivery of an intact chromosome.
Figure 1. FISH analyses. A) Untransfected control hMSCs (i) and ACE-modified hMSCs (ii) were incubated with colchicine, fixed to microscope slides, and hybridized to digoxigenin-labeled mouse major satellite DNA (red signal). Note that the ACE is intact and is maintained as an autonomous chromosome. B) ACE-modified hMSCs were induced to differentiate into either osteocytes (i) or adipocytes (ii) analysis done with interphase cells. Cells were fixed and hybridized to digoxigenin-labeled mouse major satellite DNA (red signal).
ACE-Transgene Expression During hMSC Differentiation
We evaluated whether the hMSCs carrying the RFP-ACEs still maintained their potential to differentiate into unique cell lineages. To do so, we elected to monitor reporter gene expression in the hMSC population as the cells differentiated along adipogenic and osteogenic lineages. These two pathways were chosen because the differentiated cells were visually distinctive, based on microscopic examination, and simple biochemical identification assays were available—staining lipids with Oil Red "O" or measuring insoluble calcium deposition to identify adipocytes or osteocytes, respectively. Initially differentiation was monitored in heterogeneous cell populations prior to enrichment of RFP-ACEs containing hMSCs (population with 11% RFP-expressing hMSCs). In this case, the RFP-ACE-transfected hMSCs appear to differentiate at a rate comparable to the untransfected controls. Greater than 95% of the cells had differentiated 2–3 weeks post-induction into either adipogenic or osteogenic lineages (visual inspection, data not shown). Furthermore, during differentiation RFP expression was maintained (visual inspection). FISH analyses were performed on terminally differentiated cultures 2–3 weeks post-induction in order to evaluate the integrity of the transferred RFP-ACEs during differentiation (Fig. 1B). The frequencies of RFP-ACEs were scored as 25% ± 7% (n = 2) in adipocytes and 6% ± 4% (n = 2) in osteocytes. These preliminary experiments indicate that the hMSCs stably maintained RFP-ACEs and concomitant RFP expression after osteogenic and adipogenic differentiation (Fig. 2A, 2B).
Figure 2. A) RFP-expressing adipocytes at 2 weeks post induction in the adipocyte differentiation pathway. Panel (i): Phase microscopic view from a representative field. Panel (ii): RFP-expressing cells from the identical microscopic field as depicted in panel (i). B) RFP-expressing osteocytes at 2 weeks post induction in the osteocyte differentiation pathway. Panel (i): Phase microscopic view from a representative field. Panel (ii): RFP-expressing cells from the identical microscopic field as depicted in panel (i).
RT-PCR analyses of selected gene markers (PPAR2, LDL, osteopontin) from induced and non-induced cultures were performed to further confirm the differentiated phenotype of the mesenchymal cultures containing the RFP-ACEs. Previous reports have utilized the expression of PPAR2 and LDL as hallmarks of adipogenic differentiation and osteopontin as a biomarker for osteogenic differentiation .
In order to detect transgene expression and monitor cytogenetic events during differentiation, the RFP-expressing hMSCs were enriched by flow cytometry 3–5 days post-transfection, and plated into three sets of wells. One sample (non-induced control) was maintained under hMSC culturing conditions, whereas parallel cultures were induced to differentiate into osteogenic and adipogenic lineages. Samples of induced cells were harvested at various times during the 3-week induction period. The cultures were analyzed by RT-PCR to measure the induced expression of lineage specific marker genes and by FISH. RT-PCRs for ?-actin transcripts were conducted as measures of RNA integrity for all culturing conditions since ?-actin expression is ubiquitous for both undifferentiated and differentiated hMSCs (top panels Fig. 3, lanes C-F). Only hMSCs induced to the adipogenic lineage expressed LDL or PPAR2 mRNA (Summary RT-PCR results , representative LDL profile Fig. 3 bottom panels I-M). RT-PCR detected expression of the LDL and PPAR2 genes in the genetically modified adipocyte cultures as early as 1 week post induction. Parallel osteogenic-induced cultures were tested for the presence of adipogenic lineage marker genes LDL or PPAR2. For all of the osteogenic-induced cultures, expression of LDL or PPAR2 genes was not detected (data not shown). Similar results showing lineage specificity were found when assessing osteogenic-induced cultures for expression of the osteogenic lineage marker, osteopontin, 13 days post-induction (Table 1). Overall, the RFP-ACE-modified hMSCs continued to express the RFP transgene and to maintain the integrity of the RFP-ACE after in vitro differentiation.
Figure 3: RT-PCR analyses using the ubiquitous cell lineage marker ?-actin (row B-F) and the specific adipose marker LPL (row I-M) from adipocyte mRNA. A ?-actin PCR product was found in all samples except for the negative PCR control. Top row: 515-bp ?-actin products. A) 100-bp ladder; B) negative control (no RNA); C) transfected hMSC (undifferentiated); D) transfected, 7-days post adipocyte induction; E) transfected, 13-days post adipocyte induction; F) nontransfected, 13-days post adipocyte induction; G) 100-bp ladder. Bottom row: 276-bp LPL products; H) 100-bp ladder; I) negative control (no RNA); J) transfected hMSC (undifferentiated); K) transfected, 7-days post adipocyte induction; L) transfected, 13-days post adipocyte induction; M) nontransfected, 13-days post adipocyte induction; N) 100-bp ladder. For summary of RT-PCR analyses refer to Table 1.
Table 1. Summary of RT-PCR of hMSCs cells
DISCUSSION
Chromos Molecular Systems, Inc. was responsible for all material and financial support for this research. We would like to thank Diane Monteith and Tom Stodola, for purifying ACEs and technical support; Harry Ledebur and Joseph Zendegui for critical discussions; and Shamila Gorjian for manuscript preparation.
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