STEM CELLS FOR LUNG DISEASESTEM CELLS FOR LUNG DISEASEBY BY

Dr- MOUSA ELSHAMLYDr- MOUSA ELSHAMLYLecturer of chest diseasesLecturer of chest diseases

Faculty of medicineFaculty of medicineAL-Azhar UniversityAL-Azhar University

October 2007October 2007

Stem Cells for Lung DiseaseStem Cells for Lung Disease

Introduction:Introduction:

The circulatory blood cells are formed in bone The circulatory blood cells are formed in bone marrow through a process called hematopoisis.marrow through a process called hematopoisis.

The bone marrow has an enormous production The bone marrow has an enormous production capacity, it is estimated that 10capacity, it is estimated that 1010 10 erythrocytes and erythrocytes and 101010 10 leukocytes are produce per hour in steady leukocytes are produce per hour in steady state.state.

While cell number are maintained within narrow While cell number are maintained within narrow limits in normal subject they can greatly amplified limits in normal subject they can greatly amplified on demand.on demand.

These huge cell numbers arise from smaller pool These huge cell numbers arise from smaller pool of hematopoietic stem cell (HSC).of hematopoietic stem cell (HSC).

Overview of Hematopoiesis and Stem Cell Function:

Hematopoietic stem cells are mostly Hematopoietic stem cells are mostly in arresting of non dividing state in arresting of non dividing state and have the capacity to self-and have the capacity to self-renewal.renewal.

Hematopoietic stem cells are Hematopoietic stem cells are multipotent and have the capacity to multipotent and have the capacity to differentiate into cells of all ten blood differentiate into cells of all ten blood lineages, erythrocytes, platelets, lineages, erythrocytes, platelets, neutrophils, eosinophils, basophils, neutrophils, eosinophils, basophils, monocytes, T and B lymphocytes, monocytes, T and B lymphocytes, natural killer cells and dendritic cells. natural killer cells and dendritic cells.

TABLE 1. GLOSSARY OF COMMONLY USED TERMSTABLE 1. GLOSSARY OF COMMONLY USED TERMS

Stem cells Stem cells Cells that have unlimited self-renewal properties Cells that have unlimited self-renewal properties and also have the ability to produce more and also have the ability to produce more differentiated daughter cells. differentiated daughter cells.

Progenitor cell Progenitor cell Cell able to divide and further differentiate . Cell able to divide and further differentiate .

Plasticity Plasticity Ability of cells to cross lineage barriers adopting Ability of cells to cross lineage barriers adopting phenotypes of other tissue phenotypes of other tissue

Transit amplifing cell Transit amplifing cell The progeny of stem cell with the ability to The progeny of stem cell with the ability to proliferate and differentiate. proliferate and differentiate.

Self renewalSelf renewal

TotipotentTotipotent

PluripotentPluripotent

MultipotentMultipotent

Unipotent Unipotent

Ability of the cell to produce at least one identical Ability of the cell to produce at least one identical daughter cell during cell division.daughter cell during cell division.

Ability to produce all cells of organism.Ability to produce all cells of organism.

Ability to produce cells of all lineages.Ability to produce cells of all lineages.

Ability to produce cells of multiple lineagesAbility to produce cells of multiple lineages

Ability to produce cells of a single lineage . Ability to produce cells of a single lineage .

What are stem cells:What are stem cells:

Stem cells are rlatively Stem cells are rlatively undifferentiated cells that can be undifferentiated cells that can be distinguished from their more distinguished from their more differentiated counterparts by virtue differentiated counterparts by virtue of their ability for unlimited self- of their ability for unlimited self- renewal.renewal.

Organ specific stem cells exit not Organ specific stem cells exit not only for rapidly dividing tissue such only for rapidly dividing tissue such as skin and gut but also neural tissue as skin and gut but also neural tissue and muscle.and muscle.

Much more surprising are the recent Much more surprising are the recent demonstration of stem cell plasticity in demonstration of stem cell plasticity in which organ specific stem cell have been which organ specific stem cell have been reported to differentiate into cells of other reported to differentiate into cells of other lineages either in vitro or after lineages either in vitro or after transplantation in vivo.transplantation in vivo.

The first reports of murine bone marrow The first reports of murine bone marrow derived cells differentiating into brain and derived cells differentiating into brain and muscle and cultured neural cells, followed muscle and cultured neural cells, followed by studies documenting bone marrow by studies documenting bone marrow stroma differentiating into brain, bone stroma differentiating into brain, bone marrow into liver and other ectodermal marrow into liver and other ectodermal tissue, neural cells into cardic muscle and tissue, neural cells into cardic muscle and muscle cells into blood.muscle cells into blood.

Sources of Stem Cells for LungSources of Stem Cells for Lung RepairRepair,, Regeneration and Remodeling:Regeneration and Remodeling:

Endogenous Lung Stem Cells:Endogenous Lung Stem Cells:

.Progenitor cells that participate in the maintenance and .Progenitor cells that participate in the maintenance and repair of injured lung epithelium have been described for repair of injured lung epithelium have been described for tracheobronchial, bronchiolar and alveolar compartmenttracheobronchial, bronchiolar and alveolar compartment

At least three distinct region have been described that At least three distinct region have been described that support populations of lung tissue stem cells, which is:support populations of lung tissue stem cells, which is:

Intercartilaginous regions of tracheobronchial airways.Intercartilaginous regions of tracheobronchial airways.

Neuro epithelial bodies (NEB).Neuro epithelial bodies (NEB).

Broncho – alveolar duct junction (BADJ).Broncho – alveolar duct junction (BADJ).

NEB

BADJCCSP

Each region harbors putative tissue stem Each region harbors putative tissue stem cells that share the common properties of a cells that share the common properties of a relatively undifferentiated phenotype. relatively undifferentiated phenotype.

A combination of immunophenotypic and A combination of immunophenotypic and cell ablative strategies has been employed cell ablative strategies has been employed to demonstrate that bronchiolar stem cell to demonstrate that bronchiolar stem cell residing within both the neuruepithelial residing within both the neuruepithelial bodies and Broncho-alveolar duct junction bodies and Broncho-alveolar duct junction microenvironment represent a clara cell microenvironment represent a clara cell secretary protein (CCSP), and that the secretary protein (CCSP), and that the Broncheoalveolar duct junction associated Broncheoalveolar duct junction associated population are dual for both clara cells population are dual for both clara cells secretary protein and prosurfacant protein secretary protein and prosurfacant protein C.C.

Type II pneumocytes are thought to act as Type II pneumocytes are thought to act as the main local endogenous stem cells of the main local endogenous stem cells of the lung parenchyma.the lung parenchyma.

Specialized Cells Derived fromSpecialized Cells Derived from Embryonic Embryonic Stem Cells :Stem Cells :

Differentiating cultures of embryonic stem cell Differentiating cultures of embryonic stem cell have been proposed as an alternative source of have been proposed as an alternative source of committed lung progenitor or stem cell.committed lung progenitor or stem cell.

A number of reports have now been describing A number of reports have now been describing conditions that favor differentiation of mouse conditions that favor differentiation of mouse embryonic stem cells into heterogonous embryonic stem cells into heterogonous population that include subsets of cells capable population that include subsets of cells capable of expressing lung epithelial cell specific marker of expressing lung epithelial cell specific marker such as surfactant protein C (SP-C) and clara cell such as surfactant protein C (SP-C) and clara cell secretory protein (CCSP). secretory protein (CCSP).

Ongoing research is looking for manipulation of Ongoing research is looking for manipulation of stem cells and specifically the use of extrinsic stem cells and specifically the use of extrinsic stem cells to augment lung repair and to improve stem cells to augment lung repair and to improve the response of lung parenchyma to injury and the response of lung parenchyma to injury and disease. disease.

Advantage of adult bone marrow stem cell overAdvantage of adult bone marrow stem cell over embryonicembryonic stem cellstem cell

1-Easy to be taken from bone marrow.1-Easy to be taken from bone marrow. There is no destruction of embryo and as such There is no destruction of embryo and as such

has met with moral and ethical objections .has met with moral and ethical objections . Use of embryonic stem cells associated with Use of embryonic stem cells associated with

potential of malignant transformation and potential of malignant transformation and immune rejection.immune rejection.

Plasticity of Adult Bone MarrowPlasticity of Adult Bone Marrow DerivedDerived Stem Cells Stem Cells ::

Bone marrow contain hematopoitic stem cells Bone marrow contain hematopoitic stem cells (HSCs) which characteristically differentiate (HSCs) which characteristically differentiate into every type of mature blood cells and into every type of mature blood cells and mesenchymal cells.mesenchymal cells.

Many studies have shown that cell derived from a Many studies have shown that cell derived from a adult bone marrow are able to produce a variety adult bone marrow are able to produce a variety of non hematopoietic cells both in vivo and in of non hematopoietic cells both in vivo and in vitro ,in vivo studies have mostly use systemic vitro ,in vivo studies have mostly use systemic infusion of bone marrow stem cells into infusion of bone marrow stem cells into recipient.recipient.

The donor cells are lineage marked and The donor cells are lineage marked and distinguished from host cells either phenotypi distinguished from host cells either phenotypi cally eg expressing green flurescent protein cally eg expressing green flurescent protein ( GFP), or genotypically eg containing the (Y ( GFP), or genotypically eg containing the (Y chromosome). chromosome).

In study don 2001 HSCs was In study don 2001 HSCs was transplanted from adult male mouse into transplanted from adult male mouse into female that has been lethally irradiated female that has been lethally irradiated to ablate the resident bone marrow.to ablate the resident bone marrow.

This single donor cell was able to This single donor cell was able to repopulate all bone marrow cell lineages repopulate all bone marrow cell lineages and also engrafted several organ.and also engrafted several organ.

Up to 20% of lung parenchyma in the Up to 20% of lung parenchyma in the donor mouse was found to contain Y donor mouse was found to contain Y chromosome which co localized with chromosome which co localized with epithelial marker. epithelial marker.

Recruitment of adult marrowRecruitment of adult marrow – – derived cells to the lung:derived cells to the lung:

Preexisting injury increases recruitment of Preexisting injury increases recruitment of adult marrowadult marrow – derived cells to the lung.– derived cells to the lung.

This suggests that chemotactic signals released This suggests that chemotactic signals released by injured or remodeling lung serve to attract by injured or remodeling lung serve to attract marrow – derived cells.marrow – derived cells.

Expression of specific adhesion molecules and Expression of specific adhesion molecules and soluble factors released by airway epithelium soluble factors released by airway epithelium mediate recruitment of mature circulating mediate recruitment of mature circulating leukocytes from bone marrow to the lung.leukocytes from bone marrow to the lung.

Prominent among these is stromal derived factor Prominent among these is stromal derived factor (SDF-13 CXCL 12). (SDF-13 CXCL 12).

HSCs and endothelial progenitor cells HSCs and endothelial progenitor cells (EPCs) express several cell surface (EPCs) express several cell surface adhesion molecules (CD 44, vascular cell adhesion molecules (CD 44, vascular cell adhesion molecules 1 and intra cellular adhesion molecules 1 and intra cellular adhesion molecule -1).adhesion molecule -1).

Adult marrow-human MSCs express cell Adult marrow-human MSCs express cell surface adhesion molecules including surface adhesion molecules including vascular cell adhesion molecule 1 and CD vascular cell adhesion molecule 1 and CD 44. 44.

In the lung a number of publication have demonstrated that bone marrow –derived cells can acquire phenotypic /or functional markers of airways and alveolar epithelium, vascular endothelium and interstitial cell types , in both mouse transplantation models and from lung tissue obtained from lung and bone marrow transplanted patient.

.

.In human lung specimen from some bone marrow transplanted recipient demonstrate chimerism for both epithelial and endothelial cells .

.Many of these report are based on demonstration of adult bone marrow –derived cells in the lung by detection of cells containing Y chromosome by fluorescence in situhybridization,

CD-45 positive Einterstetial tissue

Y chromosome

Effects of Stem Cells EngraftmentEffects of Stem Cells Engraftment in in TheThe Lung:Lung:

Two studies attempted to show that the normal Two studies attempted to show that the normal repair of lung relied on repair by circulating stem repair of lung relied on repair by circulating stem cells from bone marrow as well as local cells from bone marrow as well as local endogenous repair process.endogenous repair process.

They showed that lung damage secondary to They showed that lung damage secondary to lipopolysaccharide or bleomycin was increased lipopolysaccharide or bleomycin was increased if the bone marrow suppressed by either if the bone marrow suppressed by either radiation or busulphan , this could be reversed if radiation or busulphan , this could be reversed if bone marrow stem cell were transplantedbone marrow stem cell were transplanted

Studies have demonstrated that if the process of Studies have demonstrated that if the process of stem cell migration was augmented lung injury stem cell migration was augmented lung injury could be reduced.could be reduced.

Mechanism of phenotypic conversion of adult Mechanism of phenotypic conversion of adult marrow derived cells to lung cells:marrow derived cells to lung cells:Adult MSCs can be induce to develop phenotypic Adult MSCs can be induce to develop phenotypic

characteristic of fibroblasts, osteoblasts characteristic of fibroblasts, osteoblasts chondrocytes or adipocytes by in vitro exposure chondrocytes or adipocytes by in vitro exposure to growth factors and cytokines.to growth factors and cytokines.

In one report co culture of human MSCs with In one report co culture of human MSCs with primary human airway epithelial cells in vitro primary human airway epithelial cells in vitro resulted in acquisition of phenotypic and resulted in acquisition of phenotypic and functional epithelial characteristics by the functional epithelial characteristics by the MSCs .MSCs .

This suggests that release of soluble mediators This suggests that release of soluble mediators by lung epithelial cells and or direct cell-cell by lung epithelial cells and or direct cell-cell contact between epithelial and mesenchymal cell contact between epithelial and mesenchymal cell can influence phenotypic conversion of MSCs to can influence phenotypic conversion of MSCs to lung epithelial cells.lung epithelial cells.

Endothelial progenitor cells have been shown to Endothelial progenitor cells have been shown to prevent progression of pulmonary arterial prevent progression of pulmonary arterial hypertension in rat model by engrafting the hypertension in rat model by engrafting the arteriolar bed.arteriolar bed.

MSCs administration immediately after exposure MSCs administration immediately after exposure to bleomycin in mice was associated with to bleomycin in mice was associated with significant reduction of bleomycin induced significant reduction of bleomycin induced inflammation and collagen deposition within the inflammation and collagen deposition within the lung. lung.

Table 2 – Commonly Used Techniques to Detect Engraftment of Stem Cells *Technique Advantages

Light immunohistochemistry Morphologic image of labeled cells

Flourescent immunohistochemistry Dual staining of donor label and marker for engrafted cellphenotype

Flow cytometry Removes problems of overlapping cell artifact

PCR or RT-PCR of tissue Sensitivity

Laser capture microdissection Removes problems of nonspecific staining and circulating cell

Confocal/deconvolution immunohistochemistry Remove problems of overlapping cell artifact .

Reporter genes under cell specific promotor Cells only labeled if change lineage .

* PCR = polymerase chain reaction RT-PCR = real time polymerase chain reaction

Therapeutic possibilitiesTherapeutic possibilities Stem cells have a multiple clinical implication in Stem cells have a multiple clinical implication in

the lung.the lung.

Treatment by cell therapy could be investigated Treatment by cell therapy could be investigated for acute disorders such as ARDS, or more for acute disorders such as ARDS, or more chronic disorders such as emphysema and lung chronic disorders such as emphysema and lung fibrosis.fibrosis.

Engraftment of stem cells can also be used for Engraftment of stem cells can also be used for genetic therapy.genetic therapy.

In mouse model, bone marrow stem cells were In mouse model, bone marrow stem cells were genetically altered with a viral vector to express genetically altered with a viral vector to express green flourescent protein( GFP).green flourescent protein( GFP).

Theses transplanted cells then engrafted in the Theses transplanted cells then engrafted in the recipient lung, differentiating into lung recipient lung, differentiating into lung epithelium while maintaining long term epithelium while maintaining long term transgenic expression.transgenic expression.

Stem cell may therefore act as genetic vectors in Stem cell may therefore act as genetic vectors in certain diseases, needing replacement of a certain diseases, needing replacement of a protein or DNA.protein or DNA.

Within respiratory medicine cystic fibrosis (CF) is Within respiratory medicine cystic fibrosis (CF) is devastating illness that may similarly benefit from devastating illness that may similarly benefit from such an approach.such an approach.

A study providing further encouragement used A study providing further encouragement used genetic transduction of bone marrow stem cell ex genetic transduction of bone marrow stem cell ex vivo from CF patient to enable them to express vivo from CF patient to enable them to express the normal CT transmembrane conductance the normal CT transmembrane conductance regulator CFTR. regulator CFTR.

These cell were then mixed in a human These cell were then mixed in a human airway epithelial culture with epithelial airway epithelial culture with epithelial cells from CF patients.cells from CF patients.

The stem cell were able to differentiate The stem cell were able to differentiate into airway epithelial cell and partially into airway epithelial cell and partially correct the defective CFTR dependent correct the defective CFTR dependent chloride current.chloride current.

In vivo,wild type bone marrow cells were In vivo,wild type bone marrow cells were able to engraft in the mice lungs which able to engraft in the mice lungs which hase defect of CFTR,and acquire epithelial hase defect of CFTR,and acquire epithelial phenotypes including expression of CFTR phenotypes including expression of CFTR messenger RNA and protein. messenger RNA and protein.

.But this was a rare event (0.01%) and unlikely .But this was a rare event (0.01%) and unlikely to be of any clinical significance as to be of any clinical significance as approximately 10 to 15% correction of defective approximately 10 to 15% correction of defective airway epithelium hase been estimated to be airway epithelium hase been estimated to be needed to allow normal chloride current , thus needed to allow normal chloride current , thus emphasizing the gap between proof of concept emphasizing the gap between proof of concept data and clinical use. data and clinical use.

specific Use for pulmonary hypertension, specific Use for pulmonary hypertension, patient with refractory pulmonary hypertension patient with refractory pulmonary hypertension are receiving autologous EPCs transfected are receiving autologous EPCs transfected with endothelial nitric oxide synthase via with endothelial nitric oxide synthase via pulmonary artery catheter.pulmonary artery catheter.

.

. Transplantation of allogeneic stem cells to reconstitute hematopoiesis in patients with either bone marrow failure or genetic disease.

.Transplantation of autologous or allogeneic stem cells to allow reconstitution of hematopoiesis in patients who might benefit from intensive radiochemotherapy for the treatment of malignant disease.

•

•Insertion of normal gene copies into genetically defective stem cells, which could then be transplanted and expressed long-term at

sufficient levels to correct genetic disease.

•Insertion of additional copies of genes that would confer chemotherapy resistance to normal stem cells, thereby allowing dose escalation for the treatment of malignant

disease .

CONCLUSIONCONCLUSIONThere is evidence that bone marrow-derived There is evidence that bone marrow-derived

stem cells are able to target areas of the stem cells are able to target areas of the body undergoing injury and contribute to body undergoing injury and contribute to repair . the exact mechanisms are not repair . the exact mechanisms are not wholly understood , and different cell wholly understood , and different cell species may have different roles in certain species may have different roles in certain situations. Nevertheless , this basic situations. Nevertheless , this basic science research is likely to open up new science research is likely to open up new and exciting avenues of pharmacologic, and exciting avenues of pharmacologic, cellular , and genetic therapy for disease cellular , and genetic therapy for disease in which there are greatly needed. in which there are greatly needed.

Future Needs and Anticipated DifficultiesFuture Needs and Anticipated Difficulties1-How are stem cells maintained within the 1-How are stem cells maintained within the

niche and what regulates their niche and what regulates their differentiation? differentiation?

2-With improvements (current and future) in 2-With improvements (current and future) in our understanding of molecular signaling our understanding of molecular signaling pathways regulating stem cell behavior, is pathways regulating stem cell behavior, is it possible to exploit this new knowledge it possible to exploit this new knowledge for pharmacologic manipulation of stem for pharmacologic manipulation of stem cells and their progeny? cells and their progeny?

3-How are the stem cell and its progeny 3-How are the stem cell and its progeny impacted by changes in the cytokine and impacted by changes in the cytokine and growth factor milieu of the chronically growth factor milieu of the chronically diseased lung?diseased lung?

4-What opportunities exist for the generation and 4-What opportunities exist for the generation and expansion of autologous cells for transplantation? expansion of autologous cells for transplantation?

5-Basic studies are needed to develop and optimize 5-Basic studies are needed to develop and optimize methods for the isolation, purification, methods for the isolation, purification, in vitroin vitro expansion, and expansion, and in vivoin vivo validation of lung stem cells. validation of lung stem cells.

6-How might cell-based therapeutic interventions be 6-How might cell-based therapeutic interventions be considered in light of known differences between considered in light of known differences between airway regions in the cellular organization and airway regions in the cellular organization and regulation of region-specific stem cells? regulation of region-specific stem cells?

7-What mechanisms govern the observed phenotypic 7-What mechanisms govern the observed phenotypic plasticity of engrafting marrow-derived cells and the plasticity of engrafting marrow-derived cells and the efficiency of their engraftment? efficiency of their engraftment?

8-How might the beneficial effects of engrafting cells 8-How might the beneficial effects of engrafting cells while avoiding the potential harmful effects associated while avoiding the potential harmful effects associated with engraftment of exogenously supplied cells within with engraftment of exogenously supplied cells within lung tissue?lung tissue?

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