Optimization of Targeted Cell Replacement Therapy: A New Approach for Lung Disease

Cell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis, although rates of engraftment need to be improved. We previously showed improved cell retention in the lung using transtracheal delivery compared to intravenous injection. Here, we optimized other parameters of cell delivery using 7-day cultured bone marrow cells (BMCs). Retention of BMC in the lung was dose-dependent. Naphthalene treatment had maximal effects on BMC retention when given 2 days before cell delivery. Naphthalene treatment of the donor amplified a CCSP+ population and increased retention efficiency in the recipient. Repeated naphthalene treatment and repeated cell delivery both resulted in greater retention. The contribution of the second cell dose was minimal suggesting that a second delivery of BMC promotes proliferation of the first. Busulfan-induced myelosuppression augmented retention of exogenous BMC by up to 20-fold. These BMC helped CCSP reconstitution. Using the optimal delivery techniques and cytokeratin-18-driven green fluorescent protein (GFP) reporter mice, we detected threefold more GFP suggesting more BMC differentiated to epithelial cells. We propose that improved engraftment in the lung will increase cell replacement and thus be a more efficient therapeutic approach for various lung diseases. Cell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis, although rates of engraftment need to be improved. We previously showed improved cell retention in the lung using transtracheal delivery compared to intravenous injection. Here, we optimized other parameters of cell delivery using 7-day cultured bone marrow cells (BMCs). Retention of BMC in the lung was dose-dependent. Naphthalene treatment had maximal effects on BMC retention when given 2 days before cell delivery. Naphthalene treatment of the donor amplified a CCSP+ population and increased retention efficiency in the recipient. Repeated naphthalene treatment and repeated cell delivery both resulted in greater retention. The contribution of the second cell dose was minimal suggesting that a second delivery of BMC promotes proliferation of the first. Busulfan-induced myelosuppression augmented retention of exogenous BMC by up to 20-fold. These BMC helped CCSP reconstitution. Using the optimal delivery techniques and cytokeratin-18-driven green fluorescent protein (GFP) reporter mice, we detected threefold more GFP suggesting more BMC differentiated to epithelial cells. We propose that improved engraftment in the lung will increase cell replacement and thus be a more efficient therapeutic approach for various lung diseases. IntroductionCell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis. It differs from traditional regenerative medicine approaches in that there are two components. As for bone marrow transplantation, the respective local endogenous stem cells must first be removed or inactivated. Following this preconditioning regimen, exogenous cells with a preferred genotype can be supplied. Cystic fibrosis would seem to be an ideal candidate disease for such an approach, where the structure of the lung is relatively intact at the time of diagnosis. Moreover, the genetic defect is functionally limited to the bronchiolar epithelium, and partial replacement of the CFTR genotype would allow significant functional changes in chloride transport.1Johnson LG Olsen JC Sarkadi B Moore KL Swanstrom R Boucher RC Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis.Nat Genet. 1992; 2: 21-25Crossref PubMed Scopus (375) Google Scholar This contrasts with the more significant hurdles facing regenerative medicine approaches to other lung diseases, such as emphysema and pulmonary fibrosis, where repair or regeneration of the epithelial, stromal, and vascular components might all be required. However, previous studies have reported low retention of the delivered cells that prevents full assessment of the function of transplanted cells. Procedures and conditions to increase retention have received little attention to date.A variety of sources of exogenous cells could be utilized for cell replacement therapy. An ideal source would be autologous to the intended recipient, to avoid rejection by the immune system. It would also be readily accessible and able to be manipulated ex vivo, for example, to undergo gene correction of the defective CFTR locus. Finally, there would need to be at least some stem or long-lasting progenitor cells within the population to allow permanent correction of the cystic fibrosis defect. There has been substantial progress in identification of the stem cell hierarchies within the airway epithelium, at least in mice.2Rawlins EL Okubo T Xue Y Brass DM Auten RL Hasegawa H et al.The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium.Cell Stem Cell. 2009; 4: 525-534Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar,3Rock JR Onaitis MW Rawlins EL Lu Y Clark CP Xue Y et al.Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc Natl Acad Sci USA. 2009; 106: 12771-12775Crossref PubMed Scopus (956) Google Scholar However, there is no easy technique at present to isolate and expand bronchial airway stem cells.4McQualter JL Brouard N Williams B Baird BN Sims-Lucas S Yuen K et al.Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction.Stem Cells. 2009; 27: 623-633Crossref PubMed Scopus (141) Google Scholar Embryonic and induced pluripotent stem cells offer great promises, but there are currently no protocols for the robust differentiation in vitro to bronchiolar epithelium. Therefore, we have focused our efforts on bone marrow–derived progenitor populations.Although admittedly controversial, available data suggest that bone marrow–derived populations can express proteins normally expressed by the lung epithelium, especially CFTR.5Albera C Polak JM Janes S Griffiths MJ et of human pulmonary by bone marrow a to PubMed Scopus Google et and of stem cells to PubMed Scopus Google T S et of bronchial and in human lung Full Text Full Text PDF PubMed Scopus Google S R et engraftment by a bone stem Full Text Full Text PDF PubMed Scopus Google H S K S et cells can to the of in Cell PubMed Scopus Google epithelial cells and of donor stem cell PubMed Scopus Google H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Lu et of a bone population of mouse airway 2009; Google Scholar As of was able to partial restoration of bone marrow of marrow CFTR S C et of cystic fibrosis in bone marrow Natl Acad Sci USA. PubMed Scopus Google Scholar of bone marrow–derived cells at this in the of cell replacement therapy The to isolate and expand bone marrow populations is and relatively These cells can be ex and gene using as a have substantial progress the of cell replacement therapy. example, we that the transtracheal delivery substantial compared to intravenous H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Scholar We have also a population of bone marrow cells with increased for both retention in the lung and of epithelial at the and functional Lu et of a bone population of mouse airway 2009; Google Scholar Here, we have a robust for retention within the We that components of the cell replacement can be with an increase of cells in the lung at a of days of as as 20-fold. We also that delivered cells might in the and this proliferation can be augmented by a second delivery of cells. BMC can also airway regeneration and or These delivered cells can in epithelial cells and this is also augmented by a second delivery. the techniques allow robust functional assessment of delivered bone endogenous lung stem or lung epithelial of BMC BMC and naphthalene treatment on cell have been to in various of the but reported are functional the has been and we have to the The of cells and the available might have an on retention. Naphthalene treatment was to Clara cells and allow for partial of endogenous cells a lung we the of the available for retention might of cells. of 7-day cultured BMC delivered our with naphthalene treatment at H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Retention was by for the Y days cell delivery. The was compared to a by of a lung with of BMC in retention of BMC was increased in a with cell Retention that the of the delivered cell at not we the of BMC on retention BMC for days resulted in an increased retention efficiency in the lung compared to but had no effects We previously reported a population of BMC that express CCSP with functional Lu et of a bone population of mouse airway 2009; Google Scholar This population is amplified by and for up to days Finally, the optimal of naphthalene treatment was Retention efficiency was optimal when delivered 2 days naphthalene treatment that to maximal Clara cell not of BMC on retention in the previously showed that the CCSP+ population in bone marrow increased naphthalene treatment of donor from to with a at 2 days Lu et of a bone population of mouse airway 2009; Google Scholar Here, we this of naphthalene CCSP 7-day from CCSP+ or cells to transtracheal delivery. This the retention efficiency of the CCSP+ BMC from would also have retention we or 7-day cultured BMC from normal and for transtracheal delivery. BMC retention efficiency was not improved by of the donor with naphthalene at or However, retention efficiency of 7-day cultured BMC from donor was increased by naphthalene treatment of the donor at or 7-day cultured or expressed CCSP population in donor not of bone bone marrow cell from showed a population of CCSP+ cells that greater when from with naphthalene 2 days to cell The was to of cells for 7-day by cell to delivered with naphthalene 2 days CCSP+ BMC retention BMC from not greater retention efficiency in the lung of 7-day BMC from had retention efficiency the BMC from by of naphthalene treatment and repeated cell dose increase retention Clara cell by naphthalene treatment increased BMC retention efficiency in the we to that could increase Clara cell and more for delivered cells. Repeated naphthalene at days and and and to the more a treatment at where cells can be naphthalene treatment at days and and and Clara cell regeneration could be Repeated naphthalene at various time increased BMC retention efficiency by up to cell was not and repeated BMC delivery was also cell delivery increased retention efficiency in the lung by up to The increased retention was more Therefore, we the of delivery by using BMC for the first or second cell treatment Retention of the second BMC dose to be more from the first dose detected in the of two retention with repeated of naphthalene and at lung for CCSP by to naphthalene treatment at days and and and resulted in a more of CCSP are to CCSP+ cells. The 7-day bone marrow cell from donor delivered to with naphthalene in various or naphthalene treatment increased BMC retention efficiency as by delivered in two or repeated dose dose at Repeated cell delivery of showed greater cell retention efficiency compared to a dose and was optimal for days and BMC delivery at days and to retention efficiency for dose Retention of the first cell dose is increased in the of a retention efficiency of the dose is by of exogenous BMC retention BMC can to by lung in vitro This that endogenous could with delivered cells and retention in the We first 7-day cultured BMC and CCSP+ cells could also to lung we has been by to the endogenous bone marrow et stem cells in repair of the Cell PubMed Scopus Google Scholar in BMC treatment increased BMC retention efficiency in a the dose of in with naphthalene treatment was for of the mice, all also BMC and had normal to lung and to BMC can in vitro to BMC cultured for days for transtracheal delivery and CCSP+ cells from 7-day cultured BMC can also to with naphthalene at and was to endogenous bone marrow at various at increased delivered BMC retention efficiency in a The was with at naphthalene Naphthalene and treatment in of the that not BMC all BMC the by of the and by bone marrow of BMC to Clara cell the contribution of BMC in lung and in or of we with the dose of in with naphthalene to BMC the days before of the could be as in The and for CCSP or and BMC delivery to of CCSP+ airway cells and the of the The CCSP+ cell was increased by in that received BMC compared to no BMC treatment to by CCSP and at before of the could be as in lung with CCSP and or by and more CCSP in that received BMC treatment compared to that received no with also to a more intact airway in are at in CCSP+ cells from and to the airway cell by of bone marrow and of delivered to increase BMC retention in increased contribution of BMC to epithelial within the a reporter mouse was as BMC The mouse on a a by the human We naphthalene and with repeated cell delivery and as an optimized treatment and compared to the previous As the optimal resulted in increased retention as by This was when by for the Finally, to retention within the we the of delivered The optimal resulted in increased and protein progress our therapeutic of cell replacement of delivered cells. of and optimal delivery. repeated cell treatment and an optimal cell replacement resulted in in the of cell retention as by for of retention by for of the reporter was increased using the cell replacement As a the of in the of is lung with by of lung and cells in a mouse lung by or optimal are and by lung with bone marrow cell and with GFP cells from and to the airway cell treatment resulted in a increase in cells. by of green fluorescent our previous we that are in the up to days naphthalene H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Scholar Here, we could donor BMC in the lung at and delivery not long-term in the on of lung epithelial cells as by of epithelial Moreover, to avoid all the of we a mouse reporter that green fluorescent protein (GFP) the of the As previously and cultured BMC from not express GFP not H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Scholar of the the of BMC to to epithelial to the of cell replacement we approaches to retention of BMC in an cell naphthalene of cell and of all increase retention of BMC delivered the We that retention increased contribution of BMC to lung by of the at the and protein the of and there has been the contribution of BMC to the lung and other S R et engraftment by a bone stem Full Text Full Text PDF PubMed Scopus Google Williams et cells as of lung PubMed Google Scholar have that there is minimal especially in the of or that the contribution is JL for of adult stem PubMed Scopus Google JL T RC stem cells in PubMed Scopus Google RC of bone marrow to lung Cell PubMed Scopus Google MW H marrow of in pulmonary Cell PubMed Scopus Google Scholar to on studies and reporter that such a contribution is although R JL et of lung from human stem PubMed Scopus Google R T restoration of cystic fibrosis lung in with adult bone PubMed Scopus Google B N of airway epithelial and by cells naphthalene in PubMed Scopus Google Scholar where the BMC a genetic are to a contribution of H et stem cells can in PubMed Scopus Google K S et treatment of the of using bone marrow cell 2009; 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This data that cell therapy for lung disease will need to in a to bone marrow transplantation, with in cell and in and in of the and and the reporter gene GFP is by the human by for of the of H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Scholar by the received in with the of by the for and the for the and of by the on marrow and as previously H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Scholar bone marrow was by the and of donor with with and using a on at a of and the was cells with The cultured for days before for in lung and BMC delivery. given an of naphthalene as previously K R of airway cell proliferation and gene naphthalene Google Scholar transtracheal delivery of with and with the of a and cells in the trachea to the the to The to of the cell to both marrow by was given by as previously et stem cells in repair of the Cell PubMed Scopus Google Scholar was given naphthalene treatment at the dose and delivered the and in was with and and for BMC in the with and a was using for the Y and The of BMC in the was by the to the and was as the gene to gene using available at The conditions and as for 2 for at for for by using the and with of the and from the as in our previous H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Scholar lung from as and of or was to lung to gene an was and GFP was using within the human and from with cells as and The at by of and for and and at the lung was with and on and and the with in for 2 at and at with CCSP or for the the with and with with for using an from with from cell and cell with for on in by using with for The positive was to of the with 7-day cultured from and as and with vitro was using from that or with naphthalene 2 days and in and removed by and of the was in the of a with of of BMC was to the of at cells in the and are as of by of or to a as was as and IntroductionCell replacement therapy is a promising approach for treatment of lung disease such as cystic fibrosis. It differs from traditional regenerative medicine approaches in that there are two components. As for bone marrow transplantation, the respective local endogenous stem cells must first be removed or inactivated. Following this preconditioning regimen, exogenous cells with a preferred genotype can be supplied. Cystic fibrosis would seem to be an ideal candidate disease for such an approach, where the structure of the lung is relatively intact at the time of diagnosis. Moreover, the genetic defect is functionally limited to the bronchiolar epithelium, and partial replacement of the CFTR genotype would allow significant functional changes in chloride transport.1Johnson LG Olsen JC Sarkadi B Moore KL Swanstrom R Boucher RC Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis.Nat Genet. 1992; 2: 21-25Crossref PubMed Scopus (375) Google Scholar This contrasts with the more significant hurdles facing regenerative medicine approaches to other lung diseases, such as emphysema and pulmonary fibrosis, where repair or regeneration of the epithelial, stromal, and vascular components might all be required. However, previous studies have reported low retention of the delivered cells that prevents full assessment of the function of transplanted cells. Procedures and conditions to increase retention have received little attention to date.A variety of sources of exogenous cells could be utilized for cell replacement therapy. An ideal source would be autologous to the intended recipient, to avoid rejection by the immune system. It would also be readily accessible and able to be manipulated ex vivo, for example, to undergo gene correction of the defective CFTR locus. Finally, there would need to be at least some stem or long-lasting progenitor cells within the population to allow permanent correction of the cystic fibrosis defect. There has been substantial progress in identification of the stem cell hierarchies within the airway epithelium, at least in mice.2Rawlins EL Okubo T Xue Y Brass DM Auten RL Hasegawa H et al.The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium.Cell Stem Cell. 2009; 4: 525-534Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar,3Rock JR Onaitis MW Rawlins EL Lu Y Clark CP Xue Y et al.Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc Natl Acad Sci USA. 2009; 106: 12771-12775Crossref PubMed Scopus (956) Google Scholar However, there is no easy technique at present to isolate and expand bronchial airway stem cells.4McQualter JL Brouard N Williams B Baird BN Sims-Lucas S Yuen K et al.Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction.Stem Cells. 2009; 27: 623-633Crossref PubMed Scopus (141) Google Scholar Embryonic and induced pluripotent stem cells offer great promises, but there are currently no protocols for the robust differentiation in vitro to bronchiolar epithelium. Therefore, we have focused our efforts on bone marrow–derived progenitor populations.Although admittedly controversial, available data suggest that bone marrow–derived populations can express proteins normally expressed by the lung epithelium, especially CFTR.5Albera C Polak JM Janes S Griffiths MJ et of human pulmonary by bone marrow a to PubMed Scopus Google et and of stem cells to PubMed Scopus Google T S et of bronchial and in human lung Full Text Full Text PDF PubMed Scopus Google S R et engraftment by a bone stem Full Text Full Text PDF PubMed Scopus Google H S K S et cells can to the of in Cell PubMed Scopus Google epithelial cells and of donor stem cell PubMed Scopus Google H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Lu et of a bone population of mouse airway 2009; Google Scholar As of was able to partial restoration of bone marrow of marrow CFTR S C et of cystic fibrosis in bone marrow Natl Acad Sci USA. PubMed Scopus Google Scholar of bone marrow–derived cells at this in the of cell replacement therapy The to isolate and expand bone marrow populations is and relatively These cells can be ex and gene using as a have substantial progress the of cell replacement therapy. example, we that the transtracheal delivery substantial compared to intravenous H DM et cell replacement with bone marrow cells for airway epithelial Cell PubMed Scopus Google Scholar We have also a population of bone marrow cells with increased for both retention in the lung and of epithelial at the and functional Lu et of a bone population of mouse airway 2009; Google Scholar Here, we have a robust for retention within the We that components of the cell replacement can be with an increase of cells in the lung at a of days of as as 20-fold. We also that delivered cells might in the and this proliferation can be augmented by a second delivery of cells. BMC can also airway regeneration and or These delivered cells can in epithelial cells and this is also augmented by a second delivery. the techniques allow robust functional assessment of delivered bone endogenous lung stem or lung epithelial cells.

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