Name: detection of residual donor erythroid progenitor cells after hematopoietic stem cell transplantation for patients with hemoglobinopathies
Uploaded: 2017-09-06T13:00:00
Description: Watch this Scientific Journal Video about Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies at JoVE.com

Dette arbejde blev støttet af Kinderkrebshilfe Regenbogen Südtirol.

1. isolation af hæmatopoietisk knoglemarv celler af multi-parameter-fluorescens-aktiveret celle sortering <ol><li>prøve farvning <ol><li>før du starter processen, de følgende punkter skal indsamles og rede: <ol><li>Gradient medier til fremstilling af mononukleære celler (GM), 50 mL konisk rør</li> <li>12 x 75 mm flow rør til farvning celler</li> <li>farvning buffer: fosfatbufferet saltopløsning (PBS) + 3% føtalt kalveserum (FCS)</li> <li>pipetter < / l Jeg > <li>FC blok og farvning antistoffe...

<ol>
	<li>Angelucci, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+transplantation+in+thalassemia+major+and+sickle+cell+disease:+indications+and+management+recommendations+from+an+international+expert+panel.">Hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel.</a> Haematologica. 99, 811-820 (2014).</li><li>Lucarelli, G., Isgro, A., Sodani, P., Gaziev, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+transplantation+in+thalassemia+and+sickle+cell+anemia.">Hematopoietic stem cell transplantation in thalassemia and sickle cell anemia.</a> Cold Spring Harb Perspect Med. 2, a011825 (2012).</li><li>Andreani, M., Testi, M., Lucarelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+chimerism+in+haemoglobinopathies:+from+risk+of+graft+rejection+to+immune+tolerance.">Mixed chimerism in haemoglobinopathies: from risk of graft rejection to immune tolerance.</a> Tissue Antigens. 83, 137-146 (2014).</li><li>Andreani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+mixed+chimerism+in+patients+transplanted+for+the+treatment+of+thalassemia.">Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia.</a> Blood. 87, 3494-3499 (1996).</li><li>Andreani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+of+ex-thalassemic+patients+with+persistent+mixed+chimerism+after+bone+marrow+transplantation.">Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation.</a> Bone Marrow Transplant. 25, 401-404 (2000).</li><li>Kumar, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilot+study+of+prophylactic+ex+vivo+costimulated+donor+leukocyte+infusion+after+reduced-intensity+conditioned+allogeneic+stem+cell+transplantation.">Pilot study of prophylactic ex vivo costimulated donor leukocyte infusion after reduced-intensity conditioned allogeneic stem cell transplantation.</a> Biol Blood Marrow Transplant. 19, 1094-1101 (2013).</li><li>Lawler, S. D., Harris, H., Millar, J., Barrett, A., Powles, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenetic+follow-up+studies+of+recipients+of+T-cell+depleted+allogeneic+bone+marrow.">Cytogenetic follow-up studies of recipients of T-cell depleted allogeneic bone marrow.</a> Br J Haematol. 65, 143-150 (1987).</li><li>McCann, S. R., Crampe, M., Molloy, K., Lawler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemopoietic+chimerism+following+stem+cell+transplantation.">Hemopoietic chimerism following stem cell transplantation.</a> Transfus Apher Sci. 32, 55-61 (2005).</li><li>Dewald, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicenter+investigation+with+interphase+fluorescence+in+situ+hybridization+using+X-+and+Y-chromosome+probes.">A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes.</a> Am J Med Genet. 76, 318-326 (1998).</li><li>Andreani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitatively+different+red+cell/nucleated+cell+chimerism+in+patients+with+long-term,+persistent+hematopoietic+mixed+chimerism+after+bone+marrow+transplantation+for+thalassemia+major+or+sickle+cell+disease.">Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease.</a> Haematologica. 96, 128-133 (2011).</li><li>Andreani, M., Testi, M., Battarra, M., Lucarelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+chimerism+between+nucleated+and+red+blood+cells+after+bone+marrow+transplantation+for+haemoglobinopathies.">Split chimerism between nucleated and red blood cells after bone marrow transplantation for haemoglobinopathies.</a> Chimerism. 2, 21-22 (2011).</li><li>Kropshofer, G., Sopper, S., Steurer, M., Schwinger, W., Crazzolara, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+management+of+mixed+chimerism+after+bone+marrow+transplant+in+beta-thalassemia+major.">Successful management of mixed chimerism after bone marrow transplant in beta-thalassemia major.</a> Am J Hematol. 91, E357-E358 (2016).</li><li>Kimpton, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+DNA+profiling+employing+multiplex+amplification+of+short+tandem+repeat+loci.">Automated DNA profiling employing multiplex amplification of short tandem repeat loci.</a> PCR Methods Appl. 3, 13-22 (1993).</li><li>Centis, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+erythroid+expansion+in+determining+the+extent+of+apoptosis+in+erythroid+precursors+in+patients+with+beta-thalassemia+major.">The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major.</a> Blood. 96, 3624-3629 (2000).</li><li>Miccio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+selection+of+genetically+modified+erythroblastic+progenitors+leads+to+long-term+correction+of+beta-thalassemia.">In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of beta-thalassemia.</a> Proc Natl Acad Sci U.S.A. 105, 10547-10552 (2008).</li></ol>

Formålet med den aktuelle undersøgelse er at give publikum en kombination af to tilgange til analyse af donor/recipient kimærisme i erythroid progenitorceller efter HSCT hos patienter behandlet for hemoglobinopathies: 1.) Fluorescens-aktiveret celle sortering af hæmatopoietisk stamceller i knoglemarven prøver efterfulgt af en analyse af korte tandem gentagelser og 2.) kolonidannende enhed vokser af knoglemarv celler, klassificering af kolonier i forskellige stamfader typer efterfulgt af en analyse af korte tandem ge...

Allogen hæmatopoietisk stamcelletransplantation (HSCT) er den eneste tilgængelige helbredende tilgang til en række medfødte genetiske sygdomme af hæmatopoietisk systemet, at opnå sygdomsfri overlevelsesrater på mere end 90%, for ellers stærkt kompromitteret og begrænset levetid patienter1. Effekten af dette vigtigt terapeutisk redskab er optimeret ved at begrænse toksiciteten af pre- og efter transplantationen regimer2, men også af interventionerne sigter mod at opretholde stabile graft funktion, som skal være talbestemt ved nøje overvågning af donor-afledte celler3,4,5.
I almindelighed, indebærer komplet kimærisme (CC) den samlede udskiftning af lymphohematopoietic til kabinen af donor-afledte celler, hvorimod udtrykket blandet kimærisme (MC) bruges når donor - og modtager-afledte celler findes samtidig i forskellige proportioner. Split kimærisme (SC) betegner sameksistensen af blandet kimærisme observeret i encellede slægter, som i erythroid rum. Hurtig bestemmelse af kimærisme status efter HSCT er kritisk, da det kan hjælpe med at identificere patienter modtagelige for sygdom tilbagefald og indlede efterfølgende immunmodulerende strategier, som donor lymfocytter infusioner eller reduktion af immunosuppressive behandlingsformer6.
Flere metoder er blevet udviklet for at overvåge engraftment efter HSCT. Isotyping af immunoglobuliner og analyse af cytogenetisk har ringe følsomhed og er begrænsede i deres evne til at detektere polymorfi7,8. Indførelsen af fluorescerende i situ hybridisering (FISH) kan øge følsomheden i kimærisme overvågning efter HSCT, men er begrænset til sex-uoverensstemmende allografts9. I øjeblikket, polymerase kædereaktion (PCR) er den mest udbredte metode til at registrere kimærisme og er baseret på konventionelle Agarosen-acrylamid gelelektroforese af variable number tandem gentagelser (VNTRs) eller korte tandem gentager (mistænkelige). Rutinemæssigt anvendes er kvantitativ PCR købedygtig opdager en meget lille del af de resterende donor celler efter HSCT. De store begrænsning af undersøgelserne hidtil er at MC opdagelse er næsten udelukkende begrænset til tilstedeværelsen af nukleeret celler i stedet for modne erythrocytter, nemlig celler at funktionelt afgørende for patienter påvirkes af hemoglobinopathies. Hos patienter med forskellige blodtyper er det værd at huske at cytofluorometric analyse er købedygtig identificere kimærisme i røde blodlegemer ved hjælp af monoklonale antistoffer rettet mod erytrocyt antigener ABO og C, c, D, E og e10 , 11. en anderledes, men meget interessant middel til vurdering af kimærisme i erythroid afstamning er kombinationen af flow cytometric sortering af erythroid ophav og udsnit af forskellige erythroid stamfader ved dyrkning i clonogenic assays, efterfulgt af en analyse af STR12. Denne tilgang er i stand til at kvantificere relative proportioner af donor-versus-modtageren kimærisme i erythroid rum og kan udnyttes i strategien for at opretholde knoglemarv graft.

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detection of residual donor erythroid progenitor cells after hematopoietic stem cell transplantation for patients with hemoglobinopathies

Tilstedeværelsen af ufuldstændige kimærisme bemærkes, i en stor del af patienterne efter knoglemarvstransplantation for thalassemia større eller seglcelleanæmi. Denne observation har enorme konsekvenser, som efterfølgende terapeutiske immunomodulation strategier kan forbedre kliniske udfald. Konventionelt, bruges polymerase kædereaktion-baseret analyse af korte tandem gentagelser til at identificere kimærisme i donor-stammer blodlegemer. Men denne metode er begrænset til nukleeret celler og kan ikke skelne mellem dissocierede encellede lineages. Vi anvendes analyse af korte tandem gentagelser til flow cytometric-sorteret hæmatopoietisk stamceller og sammenlignede dette med analyse af korte tandem gentagelser fremstillet af udvalgte burst-dannende enhed - erythroid kolonier, både indsamlet fra knoglen marv. Med denne metode er vi i stand til at demonstrere forskellige spredning og differentiering af donor celler i erythroid rum. Denne teknik er berettiget til at fuldføre nuværende overvågning af kimærisme i stamcelletransplantation indstilling og dermed kan blive anvendt i fremtidige kliniske studier, forskning i stamceller og design af gene therapy forsøg.

Adskillelse af lymphohematopoietic ophav af FACS celle sortering
Her viser vi resultater fra sortering de nødvendige cellepopulationer for downstream STR analyse. Knoglemarv celler var plettet med V450-konjugerede anti-CD45, FITC-konjugeret anti-CD36 og APC-konjugerede anti-CD34. Befolkningens interesse er de megakaryocyte erythroid ophav (MEP), nukleeret celler ansvarlig for udviklingen af erytrocytter. Disse ...

Watch this Scientific Journal Video about Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies at JoVE.com

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

Kvantificering af donor-afledte celler er forpligtet til at overvåge engraftment efter stamcelletransplantation hos patienter med hemoglobinopathies. En kombination af flow flowcytometri-baserede celle sortering, koloni dannelse assay og efterfølgende analyse af korte tandem gentagelser kan bruges til at vurdere spredning og differentiering af stamfaderen i erythroid rum.

Påvisning af resterende Donor Erythroid stamceller efter hæmatopoietisk stamcelletransplantation til patienter med Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Research

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Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Afledning af Cardiac progenitorceller af embryonale stamceller

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Direkte Induktion af humane neurale stamceller fra perifert blod hæmatopoietiske stamceller

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Competitive Transplants to Evaluate Hematopoietic Stem Cell Fitness

Konkurrencedygtige Transplantationer at vurdere hæmatopoietisk stamcelletransplantation Fitness

The gold standard definition of a hematopoietic stem cell (HSC) is a cell that when transferred into an irradiated recipient will have the ability to ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

En ny mammedisk fedtpudstransplantationsteknik til visualisering af fartøjsgenerering af vaskulære endotelceller

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Udvikling af et In Vitro Assay at kvantificere hæmatopoietisk stilk og stamfader celler (HSPCs) i at udvikle zebrafisk embryoner

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Single-cell Photoconversion in Living Intact Zebrafish

Encellede Photoconversion i levende intakt zebrafisk

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Brug af hæmatopoietisk stamcelletransplantation at vurdere oprindelsen af myelodysplastisk syndrom

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Effektiv differentiering af humane pluripotente stamceller i leverceller

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

I Vitro Generation af Somite derivater fra menneskelige induceret celler pluripotente stamceller

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Kontrolleret kortikal indvirkning model af mus hjerneskade med terapeutisk transplantation af menneskeskabte pluripotente stamcelle-afledte neurale celler

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...