<em>In vivo</em> Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Daniela Malide; Jean-Yves M&#233;tais; Cynthia E. Dunbar

doi:10.3791/51669

In vivo clonale Monitoraggio di staminali ematopoietiche e cellule progenitrici Contrass...

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In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo clonale Monitoraggio di staminali ematopoietiche e cellule progenitrici Contrassegnato da cinque proteine fluorescenti che utilizzano confocale e microscopia Multiphoton

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17:08 min

August 6, 2014

DOI: 10.3791/51669-v

Daniela Malide¹, Jean-Yves Métais², Cynthia E. Dunbar²

¹Light Microscopy Core Facility,NHLBI/NIH, ²Hematology Branch,NHLBI/NIH

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Overview

This study presents a novel method for tracking hematopoietic stem and progenitor cells (HSPCs) in vivo using combinatorial fluorescent protein marking. By employing confocal and two-photon microscopy, researchers gain insights into the bone marrow architecture during regeneration.

Key Study Components

Area of Science

Hematopoiesis
Microscopy Techniques
Stem Cell Research

Background

Hematopoietic stem and progenitor cells play a crucial role in blood formation.
Tracking these cells in vivo can provide insights into their behavior and fate.
Traditional methods may not offer the resolution needed for detailed studies.
This study utilizes advanced imaging techniques to overcome these limitations.

Purpose of Study

To develop a method for non-invasive tracking of HSPCs in live tissues.
To investigate the clonal evolution of hematopoietic cells over time.
To enhance understanding of bone marrow-derived cell behavior in other organs.

Methods Used

Isolation and co-transduction of HSPCs with lentiviral vectors encoding five fluorescent proteins.
Transplantation of transduced cells into myeloablated recipient mice.
Harvesting of bone marrow and organs after up to 120 days.
Combination of confocal and two-photon microscopy for imaging and 3D reconstruction.

Main Results

Successful tracking of fluorescently marked HSPC-derived cells in vivo.
Visualization of clonal evolution and spatial distribution of cells over time.
Enhanced understanding of hematopoietic architecture during regeneration.
Demonstration of the advantages of high-resolution imaging techniques.

Conclusions

The method allows for detailed non-invasive studies of HSPCs in live tissues.
It provides valuable insights into hematopoiesis and cell fate mapping.
This approach can be applied to further research in stem cell biology.

Frequently Asked Questions

What are hematopoietic stem and progenitor cells?

Hematopoietic stem and progenitor cells are the cells responsible for the formation of blood cells in the body.

How does the imaging technique work?

The technique combines confocal and two-photon microscopy to visualize and track fluorescently marked cells in live tissues.

What is the significance of clonal tracking?

Clonal tracking allows researchers to study the behavior and fate of specific cell lineages over time.

What advantages does this method offer over traditional techniques?

This method provides high-resolution imaging and the ability to visualize live tissues without physical sectioning.

How long can cells be tracked using this method?

Cells can be tracked for extensive periods, up to 120 days post-transplantation.

Combinatorie 5 proteine fluorescenti marcatura di cellule staminali e progenitrici ematopoietiche consente il monitoraggio in vivo clonale tramite confocale e microscopia a due fotoni, fornendo approfondimenti midollo osseo emopoietico architettura durante la rigenerazione. Questo metodo permette di mappatura destino non invasiva di cellule HSPCs derivate spettralmente-codificati nei tessuti intatti per lunghi periodi di tempo dopo il trapianto.

L'obiettivo generale di questa procedura è quello di tracciare cloni ematopoietici marcati in fluorescenza in tessuti vivi, compreso il midollo osseo, utilizzando una nuova strategia di microscopia confocale e a due fotoni. Per fare ciò, le cellule progenitrici ematopoietiche e staminali murn vengono isolate e co-trasdotte con l'ontologia genica lentivirale o con vettori Lego che codificano per cinque proteine fluorescenti, Ian, EGFP, Venus, pomodoro TD e ciliegia m. Le cellule trasdotte vengono quindi trapiantate in topi riceventi mieloablati e dopo periodi di tempo fino a 120 giorni, il midollo osseo e gli organi vengono prelevati dai topi riceventi e viene eseguita la microscopia confocale e a due fotoni.

Le immagini risultanti vengono ricostruite in 3D per tracciare la posizione dei cloni e delle cellule trapiantati con marcatura fluorescente nel tempo. Ciò facilita lo studio dell'evoluzione clonale durante l'emopoiesi e il destino delle cellule derivate dal midollo osseo in altri organi. Il vantaggio principale di questa tecnica rispetto al sezionamento fisico dei tessuti è che combina i vantaggi dell'imaging ad alta risoluzione con il sezionamento ottico tramite microscopia confocale e la visualizzazione dei componenti strutturali tramite doppia microscopia.

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