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MESENCHYMAL STEM- AND STROMAL CELLS IN BONE MARROW AND OSTEOSARCOMA

Brune, Jan Claas LU (2011) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2011:22.
Abstract (Swedish)
Popular Abstract in Swedish

Mesenkymala stamceller (MSC) kan isoleras från en rad olika vävnader

i kroppen, bland annat från benmärg. De kan differentiera till ben, brosk,

fett- och bindvävnad men deras exakt roll i kroppen är inte väl känd.

Den senaste forskningen tyder dock på att en av deras huvuduppgifter är

att stödja blodbildningen som sker av blod- eller hematopoetiska

stamceller. Man antar att MSC bildar den hematopoetiska stamcellens

omgivning (eller niche) och därifrån stödjer blodbildningen.

MSC är väldigt enkla att isolera från kroppen och odlar man dem i

inkubator så växer de snabbt och behåller sin differentieringsförmåga.... (More)
Popular Abstract in Swedish

Mesenkymala stamceller (MSC) kan isoleras från en rad olika vävnader

i kroppen, bland annat från benmärg. De kan differentiera till ben, brosk,

fett- och bindvävnad men deras exakt roll i kroppen är inte väl känd.

Den senaste forskningen tyder dock på att en av deras huvuduppgifter är

att stödja blodbildningen som sker av blod- eller hematopoetiska

stamceller. Man antar att MSC bildar den hematopoetiska stamcellens

omgivning (eller niche) och därifrån stödjer blodbildningen.

MSC är väldigt enkla att isolera från kroppen och odlar man dem i

inkubator så växer de snabbt och behåller sin differentieringsförmåga.

Bland annat är det differentieringspotentialen som gör att dessa celler är

intressanta kandidater för kliniska användningsområden (t.ex.

transplantation). Inom denna ram är det viktigt att samla information om

dessa celler och karakterisera cellerna såväl i deras naturliga omgivning

(kroppen) som i inkubatorn. Detta var målen med delarbete (I) och

delarbete (II).



Inom (I) kunde vi visa att man med hjälp av cellmarkörer (nämligen

CD271, CD45 och CD146) kan isolera en renare stamcellspopulation än

man tidigare kunnat. Dessutom kunde vi visa att MSC i benmärgen finns

i olika omgivningar, att dessa omgivningar möjligtvis kan relatera till

olika typer av hematopoetiska stamceller och att dessa omgivningar kan

skiljas åt med hjälp av en cellmarkör som heter CD146.



I delarbete (II) analyserade vi odlade mesenkymala stamceller och

hittade en subpopulation som hade en mindre växt- och

differentieringsförmåga än huvudpopulationen vilket kan vara viktigt när

man planerar att transplantera cellerna.

Mesenkymala stamceller verkar inte bara inneha en stödjande roll vid

blodbildning men också vid tillväxt och spridning av tumörer. För

delarbete (III) valde vi att analysera en speciell typ av bencancer

(osteosarkom) där målet var att isolera och karakterisera eventuellt

förekommande MSC. Vi visade att det finns MSC i osteosarkom och att

dessa celler var väldigt lika MSC isolerade från frisk benmärg.



Målet med delarbete (IV) var att identifiera eventuella skillnader mellan

mesenkymala stamceller från bencancer och mesenkymala stamceller

isolerade från frisk vävnad.



Vi kunde visa att det inte bara fanns likheter mellan MSC från

osteosarkom och MSC från benmärg utan också skillnader: Vissa cell

markörer (som t.ex. CD142, LY6H och OSS3) förekom bara på MSC

från osteosarkom. Förhoppningsvis kommer dessa skillnader att kunna

användas som utgångspunkt för nya terapier där målet är att kapa det

stöd som de tumörstödjande mesenkymala stamcellerna ger. Preliminära

analyser kring detta utförs i skrivande stund.



Popular Abstract in German

Mesenchymale Stammzellen (MSZ) können aus einer Reihe

verschiedener Gewebe des menschlichen Körpers isoliert werden und sie

sind in der Lage zu Knochen, Knorpel, Fett- und Bindegewebe zu

differenzieren. Über die genaue Funktion dieser Zellen im Körper ist

noch nicht viel bekannt. Neueste Forschungsergebnisse deuten jedoch

darauf hin, dass die Unterstützung der Blutbildung eine ihrer

Hauptaufgaben darstellt. Blut wird von Blut- oder Hämatopoetischen

Stammzellen (HSZ) gebildet und man nimmt an, dass MSZ die direkte

Umgebung der HSZ bilden (deren Nische) und von hier die Blutbildung

unterstützen.



MSZ lassen sich verhältnismässig leicht aus Gewebeproben eines

Spenders isolieren. In Kultur ausserhalb des Körpers wachsen die Zellen

schnell und behalten ihr Differenzierungspotential. Dieses

Differenzierungspotential ist es unter anderem, was die Zellen zu

interessanten Kandidaten für klinische Anwendungsbereiche (z. Bsp.:

Transplantation) macht. Vor diesem Hintergrund ist es wichtig diese

Zellen besser zu charakterisieren, sowohl in ihrer natürlichen Umgebung

(im Körper) als auch in Kultur im Inkubator. Dieses Ziel wurde in den

Arbeiten (I) und (II) verfolgt.



In Studie (I) konnten wir zeigen, dass man mit Hilfe einer Gruppe von

Oberflächenmolekülen (CD271, CD45 und CD146) eine Stammzell-

population isolieren kann, die wesentlich höher aufgereinigt ist, als es

bisher möglich war. Darüber hinaus konnten wir zeigen, dass sich MSZ

im Knochenmark an unterschiedlichen Positionen befinden, und das die

Ursprungsposition einer Zelle im Zusammenhang mit dem

Vorhandensein des Oberflächenmoleküls CD146 steht.



In Arbeit (II) analysierten wir kultivierte MSZ und konnten nachweisen,

dass sich in den Kulturen Subpopulationen befinden, die ein geringeres

Wachstums- und Differenzierungspotential besitzen, als der Rest der

Zellen. Diese Information könnte bei der therapeutischen Anwendung

(Transplantation) kultivierter Zellen von Nutzen sein.



Mesenchymale Stammzellen scheinen allerdings nicht nur die

Blutbildung zu unterstützen, sondern auch die Entstehung und

Ausbreitung von Tumoren. Aus diesem Grund analysierten wir in Studie

(III) einen häufig vorkommenden Knochentumor (Osteosarkom) mit

dem Ziel eventuell vorkommende MSZ zu isolieren und zu

charakterisieren. Wir konnten zeigen, dass MSZ in hoher Zahl im

Osteosarkom vorkommen und dass diese MSZ jenen, aus dem

Knochenmark isolierten, sehr ähnlich sind.



Das Ziel von Arbeit (IV) war es schliesslich möglicherweise

vorhandene Unterschiede zwischen MSZ aus gesundem Knochenmark

und MSZ aus Tumoren zu identifizieren. Unsere Daten zeigen, dass

nicht nur Ähnlichkeiten bestanden sondern auch Unterschiede. Einige

Oberflächenmoleküle (CD142, LY6H und OSS3) befanden sich

beispielsweise hauptsächlich auf MSZ die aus einem Osteosarkom

isoliert worden sind. Diese Unterschiede können hoffentlich den

Ausgangspunkt für neue Krebstherapien bilden, mit dem Ziel die

Tumorunterstützung durch mesenchymale Stammzellen zu unterbrechen

und damit das Wachstum und die Ausbreitung des Tumors zu

verlangsamen. (Less)
Abstract
Primary mesenchymal stem cells (MSC) play a central role in bone marrow (BM) and during haematopoiesis. Yet, the exact phenotype and spatial distribution of primary MSC in the human BM are unknown. Their cultured progeny are promising candidates for clinical applications. MSC cultures however, are heterogeneous and, while critical for clinical applications, their exact composition is not known.

We have therefore analysed the phenotype of primary MSC in the BM and found that mesenchymal progenitors were highly enriched in the lin-/CD271+/CD45- fraction. Interestingly, CD146 expression pertained to the in-vivo localization of primary MSC in the human bone marrow (perivascular/endosteal) while CD146 expression in-vitro was oxygen... (More)
Primary mesenchymal stem cells (MSC) play a central role in bone marrow (BM) and during haematopoiesis. Yet, the exact phenotype and spatial distribution of primary MSC in the human BM are unknown. Their cultured progeny are promising candidates for clinical applications. MSC cultures however, are heterogeneous and, while critical for clinical applications, their exact composition is not known.

We have therefore analysed the phenotype of primary MSC in the BM and found that mesenchymal progenitors were highly enriched in the lin-/CD271+/CD45- fraction. Interestingly, CD146 expression pertained to the in-vivo localization of primary MSC in the human bone marrow (perivascular/endosteal) while CD146 expression in-vitro was oxygen level dependent. Cultured MSC were analysed after carboxyfluorescein succinimidyl ester (CFSE) staining for cell division tracking. Sorting for slowly dividing and rapidly dividing sub-populations and global gene expression analysis yielded 102 differentially expressed genes. Two of these genes translated into proteins that enabled for the prospective identification of a VCAM+/ FMOD+ sub-population, with low progenitor activity and a limited differentiation potential.

On the other hand, MSC support tumour growth and metastasis and have even been suggested as osteosarcoma (OS) stem cells. We therefore analysed MSC in OS (OS-MSC) and compared them with BM-MSC. OS samples contained very high frequencies of mesenchymal progenitor cells. OS-derived MSC (OS-MSC) did not show chromosomal aberrations, had normal MSC morphology and expressed the typical MSC surface marker profile. A global gene expression analysis yielded a set of genes differentially expressed between OS- and BM-MSC. Of these, 3 genes responsible for membrane-associated proteins were analysed: CD142, LY6H, and OSS3* were 24.9- , 7.2- , and 66.4-fold higher expressed in OS-MSC. The OSS3-protein was expressed in all analysed primary OS samples and only OSS3 identified the majority of mesenchymal progenitor cells in uncultured tumour samples.

Taken together, we propose here a marker combination for a highly enriched primary MSC population and show that CD146 expression relates to the perivascular (versus endosteal) localization of primary human MSC. This is of importance to further studies of the haematopoietic environment. We also show the presence of sub-populations within MSC cultures and propose markers for the isolation of a functionally impaired population. This is important for safe and efficient clinical application of cultured MSC. Furthermore we could demonstrate high numbers of colony forming mesenchymal progenitors in OS, suggesting that MSC are a major constituent of the OS stroma and consequently represent a target for therapy. Finally the above data show that a sub-population of OS cells expresses OSS3 and we furthermore demonstrate that OSS 3 identifies the majority of colony forming mesenchymal progenitors within OS. Presumably, these cells are the origin of a considerable part of the CAF population within the tumour and they should therefore be considered a target for therapy. Experiments investigating the use of OSS3 antibodies in an antibody-dependent cell-mediated cytotoxicity-based approach are currently underway.

*Due to a possible patent application, we were advised to substitute the original gene name with an alias. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • MD, PhD Östman, Arne, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
mesenchymal stem cells, stromal cells, bone marrow stem cells, tumour stroma, stroma-directed anti-tumour therapy, osteosarcoma
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2011:22
pages
146 pages
publisher
Lund University
defense location
BMC building D, floor 15 (BMC-D15) Room: Belfrage-Salen, Klinikgatan 32, 22184 Lund, Sweden
defense date
2011-03-25 13:00
ISSN
1652-8220
ISBN
978-91-86671-71-6
language
English
LU publication?
yes
id
32e4c3c7-9ae4-4a90-8882-ef8293fac413 (old id 1834853)
date added to LUP
2011-03-09 09:52:27
date last changed
2016-09-19 08:44:46
@phdthesis{32e4c3c7-9ae4-4a90-8882-ef8293fac413,
  abstract     = {Primary mesenchymal stem cells (MSC) play a central role in bone marrow (BM) and during haematopoiesis. Yet, the exact phenotype and spatial distribution of primary MSC in the human BM are unknown. Their cultured progeny are promising candidates for clinical applications. MSC cultures however, are heterogeneous and, while critical for clinical applications, their exact composition is not known. <br/><br>
We have therefore analysed the phenotype of primary MSC in the BM and found that mesenchymal progenitors were highly enriched in the lin-/CD271+/CD45- fraction. Interestingly, CD146 expression pertained to the in-vivo localization of primary MSC in the human bone marrow (perivascular/endosteal) while CD146 expression in-vitro was oxygen level dependent. Cultured MSC were analysed after carboxyfluorescein succinimidyl ester (CFSE) staining for cell division tracking. Sorting for slowly dividing and rapidly dividing sub-populations and global gene expression analysis yielded 102 differentially expressed genes. Two of these genes translated into proteins that enabled for the prospective identification of a VCAM+/ FMOD+ sub-population, with low progenitor activity and a limited differentiation potential.<br/><br>
On the other hand, MSC support tumour growth and metastasis and have even been suggested as osteosarcoma (OS) stem cells. We therefore analysed MSC in OS (OS-MSC) and compared them with BM-MSC. OS samples contained very high frequencies of mesenchymal progenitor cells. OS-derived MSC (OS-MSC) did not show chromosomal aberrations, had normal MSC morphology and expressed the typical MSC surface marker profile. A global gene expression analysis yielded a set of genes differentially expressed between OS- and BM-MSC. Of these, 3 genes responsible for membrane-associated proteins were analysed: CD142, LY6H, and OSS3* were 24.9- , 7.2- , and 66.4-fold higher expressed in OS-MSC. The OSS3-protein was expressed in all analysed primary OS samples and only OSS3 identified the majority of mesenchymal progenitor cells in uncultured tumour samples.<br/><br>
Taken together, we propose here a marker combination for a highly enriched primary MSC population and show that CD146 expression relates to the perivascular (versus endosteal) localization of primary human MSC. This is of importance to further studies of the haematopoietic environment. We also show the presence of sub-populations within MSC cultures and propose markers for the isolation of a functionally impaired population. This is important for safe and efficient clinical application of cultured MSC. Furthermore we could demonstrate high numbers of colony forming mesenchymal progenitors in OS, suggesting that MSC are a major constituent of the OS stroma and consequently represent a target for therapy. Finally the above data show that a sub-population of OS cells expresses OSS3 and we furthermore demonstrate that OSS 3 identifies the majority of colony forming mesenchymal progenitors within OS. Presumably, these cells are the origin of a considerable part of the CAF population within the tumour and they should therefore be considered a target for therapy. Experiments investigating the use of OSS3 antibodies in an antibody-dependent cell-mediated cytotoxicity-based approach are currently underway. <br/><br>
 *Due to a possible patent application, we were advised to substitute the original gene name with an alias.},
  author       = {Brune, Jan Claas},
  isbn         = {978-91-86671-71-6},
  issn         = {1652-8220},
  keyword      = {mesenchymal stem cells,stromal cells,bone marrow stem cells,tumour stroma,stroma-directed anti-tumour 
therapy,osteosarcoma},
  language     = {eng},
  pages        = {146},
  publisher    = {Lund University},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {MESENCHYMAL STEM- AND STROMAL CELLS IN BONE MARROW AND OSTEOSARCOMA},
  volume       = {2011:22},
  year         = {2011},
}