Derivation of lung epithelium from human cord blood-derived mesenchymal stem cells

Abstract

Rationale: Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases.

Objectives: We hypothesized that human umbilical cord blood stem cells might be an effective alternative to adult bone marrow and embryonic stem cells for regeneration and repair of injured airway epithelium.

Methods: Human cord blood was obtained from normal deliveries at the University of Vermont. Cultured plastic adherent cells were characterized as mesenchymal stem cells (MSCs) by flow cytometry and differentiation assays. Cord blood-derived MSCs (CB-MSCs) were cultured in specialized airway growth media or with specific growth factors, including keratinocyte growth factor and retinoic acid. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. CB-MSCs were systematically administered to immunotolerant, nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice. Lungs were analyzed for presence of human cells.

Measurements and main results: When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed Clara cell secretory protein (CCSP), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein C, and thyroid transcription factor-1 mRNA, and CCSP and CFTR protein. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression.

Conclusions: CB-MSCs appear to be comparable to MSCs obtained from adult bone marrow in ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo.

Figures

Figure 1.

Morphologies of cord blood mononuclear cells (CB-MNCs) and cord blood mesenchymal stem cells (CB-MSCs). (A) Passage 0 cultures demonstrate an initial mixed cell population; (B) CB-MSCs after one passage showed uniform fibroblastic-like morphology. Standard phase contrast microscopy. Original magnification, ×10 and filter Ph1.

Figure 2.

(A) Cord blood (CB) plastic adherent cells express CD73, CD105, and CD90, but not CD34 and CD45. (B) CB plastic adherent cells between passage 2 and 4 express cell surface markers that meet the International Society for Cellular Therapy criteria for mesenchymal stem cells (21).

Figure 3.

Cord blood mesenchymal stem cells (CB-MSCs) can differentiate into osteoblast, adipocytes, and chondroblasts in vitro. CB-MSCs were incubated to confluency in CB basal medium and then cultured in CB basal medium or in different induction media for 21 days. (A–C) Phase contrast micrograph of CB-MSCs cultured in (A) CB basal medium and stained with Alizarin Red S and Oil Red O, (B) osteogenic medium for 21 days and stained with Alizarin Red S for calcium deposits (shown in red), or (C) adipogenic medium and stained with Oil Red O for lipid droplets (shown in red). Chondrogenesis (D) was produced by incubating 200,000 CB-MSCs as a micromass pellet in chrondrogenic media for 21 days and 5-μm paraffin-embedded sections were stained with Toluidine blue for histologic assessment. Photomicrographs demonstrate representative findings from passage 4 CB-MSCs. Original magnification, ×20, except inset, ×40. Specific constituents of each media formulation are included in the online supplement Methods.

Figure 4.

Cord blood mesenchymal stem cells (CB-MSCs) express Clara cell secretory protein (CCSP) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA when cultured in specialized airway growth medium. (A) Representative reverse transcriptase–polymerase chain reaction (RT-PCR) gel demonstrating expression of lung epithelial markers under different culture conditions (40 cycles RT-PCR). (B) Quantitative PCR confirming CCSP and CFTR mRNA expression in CB-MSCs cultured in MTEC medium and SAGM compared with cells maintained in CB basal medium. Bars represent means ± SE of five samples cultured in either MTEC medium or in SAGM. (C) RT-PCR gels demonstrating expression of fibroblast and myofibroblast markers under different culture conditions (40 cycles RT-PCR). Data represent representative findings from one of eight similar experiments using different CB preparations. CB-MNC = cord blood mononuclear cells; HL = human lung; HLF = human lung fibroblast; HLF-no RT = human lung fibroblast and no reverse transcriptase; KGF = keratinocyte growth factor; MTEC = mouse tracheal epithelial cells; RA = retinoic acid; SAGM = small airway growth medium.

Figure 4.

Cord blood mesenchymal stem cells (CB-MSCs) express Clara cell secretory protein (CCSP) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA when cultured in specialized airway growth medium. (A) Representative reverse transcriptase–polymerase chain reaction (RT-PCR) gel demonstrating expression of lung epithelial markers under different culture conditions (40 cycles RT-PCR). (B) Quantitative PCR confirming CCSP and CFTR mRNA expression in CB-MSCs cultured in MTEC medium and SAGM compared with cells maintained in CB basal medium. Bars represent means ± SE of five samples cultured in either MTEC medium or in SAGM. (C) RT-PCR gels demonstrating expression of fibroblast and myofibroblast markers under different culture conditions (40 cycles RT-PCR). Data represent representative findings from one of eight similar experiments using different CB preparations. CB-MNC = cord blood mononuclear cells; HL = human lung; HLF = human lung fibroblast; HLF-no RT = human lung fibroblast and no reverse transcriptase; KGF = keratinocyte growth factor; MTEC = mouse tracheal epithelial cells; RA = retinoic acid; SAGM = small airway growth medium.

Figure 4.

Cord blood mesenchymal stem cells (CB-MSCs) express Clara cell secretory protein (CCSP) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA when cultured in specialized airway growth medium. (A) Representative reverse transcriptase–polymerase chain reaction (RT-PCR) gel demonstrating expression of lung epithelial markers under different culture conditions (40 cycles RT-PCR). (B) Quantitative PCR confirming CCSP and CFTR mRNA expression in CB-MSCs cultured in MTEC medium and SAGM compared with cells maintained in CB basal medium. Bars represent means ± SE of five samples cultured in either MTEC medium or in SAGM. (C) RT-PCR gels demonstrating expression of fibroblast and myofibroblast markers under different culture conditions (40 cycles RT-PCR). Data represent representative findings from one of eight similar experiments using different CB preparations. CB-MNC = cord blood mononuclear cells; HL = human lung; HLF = human lung fibroblast; HLF-no RT = human lung fibroblast and no reverse transcriptase; KGF = keratinocyte growth factor; MTEC = mouse tracheal epithelial cells; RA = retinoic acid; SAGM = small airway growth medium.

Figure 5.

Cord blood mesenchymal stem cells (CB-MSCs) express Clara cell secretory protein (CCSP) and cystic fibrosis transmembrane conductance regulator (CFTR) protein after culture in specialized airway growth medium (MTEC medium and SAGM). Representative immunofluorescent staining photomicrography of (A) CB-MSCs cultured in CB basal medium, (B) CB-MSCs cultured in MTEC medium, (C) CB-MSCs cultured in SAGM, inset shows a higher magnification of a cell and (D) human bronchial epithelial cells (positive control inset shows a higher magnification of a cell). Pink arrows indicate cells that do not express either CCSP or CFTR, white arrows indicate cells that express only CCSP but not CFTR, and yellow arrows indicate cells that express both CCSP and CFTR. Blue = DAPI (4′-6-diamidino-2-phenylindole) nuclear stain, green = CCSP, red = CFTR, orange = costaining of CCSP and CFTR. Original magnification, ×40. Images were adjusted for brightness and contrast with Photoshop version 6.0 (Adobe Systems, San Jose, CA). Data represent representative findings from one of eight similar experiments using different CB preparations.

Figure 6.

Cord blood mesenchymal stem cells (CB-MSCs) can be transduced with recombinant lentivirus yellow fluorescent protein (YFP) and cystic fibrosis transmembrane conductance regulator (CFTR). Phase contrast (A, C, E) and fluorescent (B, D, F) microscopy of CB-MSCs expressing either YFP (A, B) or CFTR (C, D) after lentiviral transduction. YFP direct fluorescence was pseudocolored to green. E and F represent nontransduced control CB-MSCs. Original magnification, ×40. Images were adjusted for brightness and contrast with Photoshop version 6.0. Data represent representative findings from one of two similar experiments using different CB preparations.

Figure 7.

Quantitative polymerase chain reaction of human Alu genomic DNA in recipient lungs. (A) Quantity of DNA (ng) detected in recipient lung. (B) Index of DNA quantity compared with control (nontransplanted) lungs. The horizontal bars represent the mean value for each experimental condition. CB = cord blood; NOD-SCID = nonobese diabetic/severe combined immunodeficiency mice.

Figure 7.

Quantitative polymerase chain reaction of human Alu genomic DNA in recipient lungs. (A) Quantity of DNA (ng) detected in recipient lung. (B) Index of DNA quantity compared with control (nontransplanted) lungs. The horizontal bars represent the mean value for each experimental condition. CB = cord blood; NOD-SCID = nonobese diabetic/severe combined immunodeficiency mice.

Figure 8.

Human β2-microglobulin– and pancytokeratin-positive cells are mostly detected in NOD-SCID mice alveolar walls after systemic administration of cord blood mesenchymal stem cells (CB-MSCs). (A–C) NOD-SCID lung sections 2 weeks, 1 month, and 3 months after CB-MSC administration, respectively. Blue = DAPI (4′-6-diamidino-2-phenylindole) nuclear stain, green = pancytokeratin, red = β2-microglobulin, white = colocalization of pancytokeratin and β2-microglobulin. Original magnification, ×200. Representative photomicrographs from one of four mice per experimental condition.

Figure 9.

Human β2-microglobulin positive cells can be detected in NOD-SCID mice airways after systemic administration of cord blood mesenchymal stem cells (CB-MSCs). (A–C) NOD-SCID lung sections 2 weeks after CB-MSC administration, (D) NOD-SCID lung section 1 month after CB-MSC administration, and (E) NOD-SCID lung section 3 months after CB-MSC administration. Blue = DAPI (4′-6-diamidino-2-phenylindole) nuclear stain, green = pancytokeratin, red = β2-microglobulin, white = colocalization of pancytokeratin and β2-microglobulin. Columns 2–5, original magnification, ×200. (See original size in Figure E2). Representative photomicrographs from one of four mice per experimental condition.

Figure 10.

Quantitative determination of donor-derived cells in recipient lungs. Bars represent means ± SD from quantitative determinations on lung sections from three mice per experimental condition. β2+ CK+ = β2-microglobulin– and pancytokeratin-positive cells.

Figure 11.

Human β2-microglobulin– and cystic fibrosis transmembrane conductance regulator (CFTR)–positive cells can be detected in NOD-SCID mice airways after systemic administration of cord blood mesenchymal stem cells (CB-MSCs). (A and B) NOD-SCID lung sections 2 weeks and 1 month after CB-MSC administration, respectively. (C and D) Corresponding higher power images of (A) and (B). Representative photomicrographs from one of four mice per experimental condition.