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Enumeration, Expansion, and Differentiationof Human Mesenchymal Progenitor Cells

Using MesenCult®

T E C H N I C A L M A N U A LV E R S I O N 2 . 2 . 0

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For Research Use Only

In North AmericaTel: 1.604.877.0713Fax: 1.604.877.0704NA Toll Free Tel: 1.800.667.0322NA Toll Free Fax: 1.800.567.2899e-mail: [email protected]: [email protected]

StemCell TechnologiesVersion 2.2.0

Oct 2007Catalog #28453

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Table of Contents1.0 Introduction .........................................................................................................................1

2.0 Materials ..............................................................................................................................2

2.1 Product Descr�pt�on ...........................................................................................................................22.2 Related Products ...............................................................................................................................22.3 Storage Cond�t�ons ............................................................................................................................22.4 Equ�pment, Suppl�es, and Reagents Requ�red ...................................................................................3

3.0 Human Colony-Forming Unit - Fibroblast (CFU-F) Assay .................................................4

3.1 Culture Set-up ...................................................................................................................................43.1.1 Preparat�on of Complete MesenCult® Med�um (Human) ........................................................43.1.2 Process�ng of Cells and the CFU-F Assay .............................................................................4

3.2 Suggested Procedure for Sta�n�ng and Enumerat�on of CFU-F-Der�ved Colon�es ...............................53.2.1 Sta�n�ng ................................................................................................................................53.2.2 Enumerat�on .........................................................................................................................5

3.3 Photographs of Human CFU-F-Der�ved Colon�es...............................................................................6

4.0 Expansion of Cultured Mesenchymal Cells ........................................................................7

4.1 Culture Set-up ...................................................................................................................................74.2 Passag�ng Cultured Mesenchymal Cells ............................................................................................74.3 Photographs of Cultured Mesenchymal Cells ....................................................................................84.4 Phenotype of Cultured Cells ..............................................................................................................9

5.0 Helpful Hints and Frequently Asked Questions ...............................................................10

5.1 Helpful H�nts ....................................................................................................................................105.2 Frequently Asked Quest�ons ............................................................................................................10

6.0 Mesenchymal Adipogenic Assay using MesenCult® .......................................................12

6.1 Culture Set-up .................................................................................................................................126.2 Photographs of Human Ad�pocytes .................................................................................................13

7.0 Mesenchymal Osteogenic Assay using MesenCult® .......................................................14

7.1 Preparat�on of Complete MesenCult® Osteogen�c Med�um ..............................................................147.2 Culture Set-up .................................................................................................................................15

8.0 Appendix 1: RosetteSep® Human Mesenchymal Stem Cell Enrichment Cocktail ........ 16

8.1 RosetteSep® Procedure (Catalog #15128/15168)............................................................................168.2 RosetteSep® Procedure Summary ...................................................................................................17

9.0 References .......................................................................................................................18

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1.0 IntroductionThe bone marrow stroma was or�g�nally thought to funct�on ma�nly as a structural framework for hematopo�et�c stem and progen�tor cells �n the bone marrow. It has now been establ�shed that the stroma cons�sts of a heterogeneous populat�on of cells �nclud�ng endothel�al cells, fibroblasts, ad�pocytes, and osteogen�c cells, a subset of wh�ch exerts both pos�t�ve and negat�ve regulatory effects on the prol�ferat�on and d�fferent�at�on of hematopo�et�c cells.1,2 The adherent stromal cell populat�on �s also bel�eved to conta�n other non-hematopo�et�c cells that are capable of both self-renewal and d�fferent�at�on �nto bone, cart�lage, muscle, tendon, and fat.3-5 Character�zat�on of the stromal cells was �n�t�ated �n the early 1980s when the morpholog�cal and cytochem�cal propert�es of the cultured cells were descr�bed (Sudan Black+, alkal�ne phosphatase+, esterase-, collagen IV+, fibronect�n+).6,7 In 1991, S�mmons and Torok-Storb descr�bed the first ant�body (STRO-1) that targeted the stromal precursor �n human bone marrow.8

The colony-form�ng un�t-fibroblast (CFU-F) assay �s used by many �nvest�gators as a funct�onal method to quant�fy stromal progen�tor cells. There appears to be a strong correlat�on between age and prol�ferat�ve potent�al, w�th decreas�ng progen�tor cell prol�ferat�on assoc�ated w�th �ncreas�ng age.9,10 Abnormal funct�on of stromal precursor cells has been �mpl�cated �n several d�seases.11,12 Transplantat�on of unprocessed bone marrow cells can restore m�cro-env�ronmental funct�on, suggest�ng that unprocessed bone marrow conta�ns both stromal precursor cells and hematopo�et�c precursor cells. Stud�es by Gallatto et al. demonstrate that these m�croenv�ronmental precursor cells, as measured by the CFU-F assay, are suscept�ble to damage follow�ng chemotherapy or rad�at�on and rema�n at a s�gn�ficantly reduced frequency for a cons�derable t�me follow�ng transplantat�on.13 Acknowledg�ng that stromal cells prov�de regulatory st�mul� to other cells may expla�n �n part why there �s a slow and skewed recovery of many �mmune cell populat�ons follow�ng transplantat�on.14

In recent years there has been �ncreased �nterest �n stromal cells and the�r funct�on �n both the t�ssue eng�neer�ng and stem cell plast�c�ty fields. Th�s �ncrease �n �nterest has been fueled by the observat�on that cultured stromal cell populat�ons are capable of both self-renewal and d�fferent�at�on, character�st�cs typ�cally assoc�ated w�th stem cells. These tra�ts have led many researchers to refer to cultured stromal cells as mesenchymal stem cells (MSC).

Horw�tz and colleagues attempted to clar�fy and standard�ze the term�nology for mesenchymal stem cells (MSC) when they proposed the term�nology “mult�potent mesenchymal stromal cells” to descr�be all cultured fibroblast-l�ke plast�c-adherent cells, reserv�ng the term “mesenchymal stem cells” for a subset of cells that clearly demonstrate stem cell propert�es.15 Horw�tz and colleagues further stated that for both cell populat�ons the acronym MSC may be used w�th deta�led descr�pt�on of the character�st�cs of the cell populat�on stud�ed by each �nvest�gator. Th�s d�st�nct�on �s �mportant �n the mesenchymal research field because mesenchymal cells exh�b�t d�fferent phenotypes depend�ng on var�ous factors such as culture method, med�um compos�t�on, plat�ng dens�ty of the cells, and source of �solated cells.

Cultured mesenchymal cells have been character�zed us�ng panels of ant�bod�es and are defined as CD45-CD34-

SH2(CD105)+SH3+CD90(THY-1)+ cells.5 Other markers used by researchers to �dent�fy these cultured mesenchymal cells �nclude STRO-1, CD49a, SB10, D7-FIB, LNGFR, CD106, HOP-26, CD144, CD166, CD115, CD29 and HLA-ABC.16-20 However, the exact phenotype of the stromal (mesenchymal) precursor cell �n human bone marrow (�.e. the cell phenotype pr�or to culture) �s st�ll debated. The �solat�on and enr�chment of human mesenchymal cells have ut�l�zed some of the s�mple character�st�cs of the human MSC, such as adherence of the cell when cultured, as well as cell separat�on strateg�es us�ng cockta�ls of ant�bod�es that deplete the bone marrow of spec�fic cell populat�ons.21,22

Cultured mesenchymal cells have been shown to exh�b�t some un�que propert�es that challenge the dogma that stem cells der�ved from adult t�ssue produce only the cell l�neages character�st�c of t�ssues �n wh�ch they res�de. Stud�es by Verfa�ll�e’s group have demonstrated the ab�l�ty of cultured MAPC, a subset of cells copur�fied w�th MSC, to d�fferent�ate �nto neural cells, skeletal cells, card�omyocytes, endothel�al cells and smooth muscle cells.23 The expand�ng knowledge of the b�ology of spec�fic cell populat�ons may be the foundat�on for future therap�es �n many areas outs�de of hematology and oncology.

There �s a great deal of cl�n�cal �nterest �n mesenchymal cells and the follow�ng cl�n�cal appl�cat�ons are presently be�ng evaluated:

• expans�on and re�nfus�on of MSC �nto pat�ents �n an attempt to reconst�tute the m�croenv�ronment and prov�de opt�mal cond�t�ons to support hematopo�es�s24

• gene transfer �nto MSC25

• repa�r of mesenchymal t�ssues26,27

• ab�l�ty of mesenchymal cells to support hematopo�et�c cell expans�on28,29

• treatment of graft versus host d�sease (GVHD)30,31

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2.0 Materials

2.1 Product Description

2.2 Related Products

2.3 Storage Conditions

MesenCult® MSC Basal Med�um (Human; Catalog #05401) �s stable at 2 - 8°C for 1 year from date of manufacture.

Mesenchymal Stem Cell St�mulatory Supplements (Human; Catalog #05402) and Ad�pogen�c St�mulatory Supplements (Human; Catalog #05403) are stable at -20°C for 2 years from date of manufacture. Storage at 2 - 8°C �s not recommended. If the ent�re volume of supplements �s not needed at one t�me, they can be al�quoted and refrozen. Do not freeze-thaw al�quots more than tw�ce.

Storage Cond�t�ons for MesenCult® Osteogen�c St�mulatory K�t (Catalog #05404) Components:

Catalog # Description Unit Size Stability and Storage Conditions

05401MesenCult® MSC Basal Med�um (Human)

450 mLStore at 2 - 8°C. Stable for 1 year from date of manufacture.

05405 Osteogen�c St�mulatory Supplements (Human)

80 mLStore at -20°C. Stable for at least 1 year.

05406 ß-Glycerophosphate 1.0 M 10 mLStore at -20°C. Stable for at least 2 years.

05407 Dexamethasone 1 mgStore at 2 - 8°C. Stable for at least 2 years.

07157 Ascorb�c Ac�d 100 mgStore at room temperature. Stable for at least 2 years.

Product Description Volume Catalog #

MesenCult® MSC Basal Med�um (Human) 450 mL 05401

Mesenchymal Stem Cell St�mulatory Supplements (Human)

50 mL 05402

MesenCult® Ad�pogen�c St�mulatory Supplements (Human)

50 mL 05403

MesenCult® Osteogen�c St�mulatory K�t (Human) To make 500 mL 05404

Product Description Volume Catalog #

Frozen Marrow Stromal Cells 7.5 x 105 cells MSC-001F

Fetal Bov�ne Serum for Human Mesenchymal Stem Cells

100 mL500 mL

0647106472

Fetal Bov�ne Serum for Human Osteogen�c Precursors

100 mL500 mL

0647306474

RosetteSep® Mesenchymal Stem Cell Enr�chment Cockta�l

2 mL10 mL

1512815168

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2.4 Equipment, Supplies, and Reagents Required

To perform all assays, the follow�ng equ�pment and reagents are requ�red, �n add�t�on to MesenCult® MSC Basal Med�um and Supplements:

• B�ohazard safety cab�net cert�fied for level II handl�ng of b�olog�cal mater�als (e.g. Canad�an Cab�nets) • 37°C �ncubator w�th hum�d�ty and gas control to ma�nta�n >95% hum�d�ty and an atmosphere of 5% CO2 �n a�r

(e.g. Forma 3326)• Laboratory centr�fuge • Standard l�ght m�croscope (for cell count�ng) • Inverted m�croscope • Hemacytometer • Good qual�ty t�ssue culture-treated d�shes, flasks, or plates: 100 mm culture d�shes (Catalog #27125/27127),

T-25 cm2 t�ssue culture flasks (Falcon Catalog #353109), or 6-well t�ssue culture-treated plates (Falcon Catalog #353502 or Corn�ng Catalog #3506)

Always use tissue culture-treated dishes, flasks, or plates to culture mesenchymal stem cells.• 1 mL and 10 mL ster�le p�pettes • P�pette-a�d (e.g. Drummond Sc�ent�fic)• 14 mL polypropylene tubes (Falcon Catalog #352001)• PBS w�th 2% Fetal Bov�ne Serum (Catalog #07905) • Ammon�um Chlor�de Solut�on (Catalog #07800) • PBS (Catalog #37350) • Tryps�n-EDTA (Catalog #07901) • F�coll-Paque™ PLUS (Catalog #07957) • 3% Acet�c Ac�d w�th Methylene Blue (Catalog #07060) • Trypan Blue (Catalog #07050)

To sta�n CFU-F colon�es, the follow�ng mater�als are requ�red:

• PBS (Catalog #37350) • Methanol ACS (BDH Catalog #ACS531) • G�emsa Sta�n�ng Solut�on (EMD Chem�cals Catalog #R03055) • D�st�lled water

The Osteogen�c Assay requ�res:

• 70 µm cell stra�ner (Falcon Catalog #352350)

F�coll-PaqueTM �s a trademark of GE HealthCare Ltd.

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3.0 Human Colony-Forming Unit - Fibroblast (CFU-F) Assay Fresh samples of human bone marrow can be processed and plated �n l�m�t�ng cell dens�t�es to assess the number of mesenchymal stem and progen�tor cells us�ng the CFU-F assay.

3.1 Culture Set-Up

3.1.1 Preparation of Complete MesenCult® Medium (Human)

Thaw Mesenchymal Stem Cell St�mulatory Supplements (Human; Catalog #05402) at room temperature or 2 - 8°C overn�ght. Add the ent�re contents of the Mesenchymal St�mulatory Supplements (Human) to MesenCult® MSC Basal Med�um (Human; Catalog #05401) and m�x thoroughly. Th�s �s now referred to as Complete MesenCult® Medium (Human).

Complete MesenCult® Med�um (Human) �s stored at 2 - 8°C and should be prepared �n volumes that can be used w�th�n 1 month. If less than 500 mL w�ll be requ�red, smaller volumes can be prepared. Prepare Complete MesenCult® Med�um (Human) by d�lut�ng Mesenchymal Stem Cell St�mulatory Supplements (Human) 1/10 w�th MesenCult® MSC Basal Med�um (Human). For example, prepare 100 mL of Complete MesenCult® Med�um (Human) by add�ng 10 mL of Mesenchymal Stem Cell St�mulatory Supplements (Human) to 90 mL of MesenCult® MSC Basal Med�um (Human).

It is not necessary to add additional fetal bovine serum to Complete MesenCult® Medium (Human). Fetal bovine serum is already a component of the Mesenchymal Stem Cell Stimulatory Supplements (Human).

For long-term storage of Complete MesenCult® Medium (Human) refer to Section 5.1: Helpful Hints.

3.1.2 Processing of Cells and the CFU-F Assay1. When work�ng w�th a fresh bone marrow (BM) sample, the cells need to be processed to remove the red blood

cells or enr�ch des�red cells pr�or to culture. Choose one of the follow�ng methods: • Ammon�um chlor�de lys�s (to remove the red blood cells, Catalog #07800) • Isolat�on of the mononuclear cells by F�coll-Paque™ PLUS (Catalog #07907/07957) dens�ty grad�ent separat�on• Enr�chment of mesenchymal stem cells us�ng the RosetteSep® Human Mesenchymal Stem Cell Enr�chment K�t

(Catalog #15128/15168, refer to Append�x 1 for more �nformat�on)

The CFU-F assay cannot be performed with previously frozen bone marrow mononuclear cells or culture-expanded mesenchymal stem cells.

2. After process�ng, wash the cells by add�ng 10 mL of PBS w�th 2% FBS (Catalog #07905) to the cell pellet. Centr�fuge the cells at 300 x g (~1200 rpm) for 10 m�nutes at 20°C. Remove the supernatant and resuspend the cells �n 1 - 2 mL of Complete MesenCult® Med�um (Human).

3. If work�ng w�th BM cells processed w�th ammon�um chlor�de or F�coll-Paque™ PLUS, perform a nucleated cell count (us�ng 3% Acet�c Ac�d w�th Methylene Blue, Catalog #07060) and d�lute the cells to a stock cell concentrat�on of 2 x 106 cells/mL �n Complete MesenCult® Med�um (Human). If work�ng w�th enr�ched mesenchymal cells �solated us�ng the RosetteSep® Enr�chment K�t for Human Mesenchymal Stem Cells (Catalog #15128/15168), perform a cell count and d�lute cells to a stock cell concentrat�on of 5 x 105 cells/mL �n Complete MesenCult® Med�um (Human).

4. Plate three d�fferent cell dens�t�es by add�ng 1.0 mL, 0.5 mL, and 0.25 mL of the cells at stock concentrat�on to separate 100 mm t�ssue culture-treated d�shes (or T-25 cm2 t�ssue culture flasks) prefilled w�th Complete MesenCult® Med�um (Human) to a total volume of 10 mL. For F�colled or lysed BM cells, th�s w�ll y�eld final cell count of 2 x 106 cells, 1 x 106 cells and 0.5 x 106 cells �n 10 mL of med�um. For RosetteSep®-enr�ched cells th�s w�ll y�eld final cell count of 5 x 105 cells, 2.5 x 105 cells and 1.25 x 105 cells �n 10 mL of med�um.

Plating three concentrations will ensure that the resulting numbers of colonies can be scored, as there are differences in the proliferative potential of CFU-F from various bone marrow samples.

5. Place the 100 mm d�shes (or T-25 cm2 t�ssue culture flasks) �nto a 37°C hum�d�fied �ncubator w�th 5% CO2 �n a�r and >95% hum�d�ty for 14 days.

Maximum colony size and numbers are typically observed at 14 days.


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3.2 Suggested Procedure for Staining and Enumeration of CFU-F-Derived Colonies

3.2.1 Staining

1. Remove the med�um from cultures of CFU-F grown �n 100 mm t�ssue culture d�shes or T-25 cm2 t�ssue culture flasks and d�scard �nto the b�ohazardous waste. The adherent colon�es w�ll rema�n attached to the plate.

2. Wash the culture d�shes or flasks tw�ce us�ng PBS (Catalog #37350) to remove any rema�n�ng med�um. D�scard the PBS from the two washes �nto the b�ohazardous waste.

3. Add 5 mL of methanol to each culture d�sh or flask for 5 m�nutes at room temperature.

Addition of methanol fixes the cells to the tissue culture dishes or flasks.

4. Remove the methanol and d�scard �nto the b�ohazardous waste. Let the culture d�shes or flasks a�r dry at room temperature.

5. Add 5 mL of G�emsa Sta�n�ng Solut�on (EMD Chem�cals Catalog #R03055) to each culture d�sh or flask and leave for 5 m�nutes.

6. Remove the G�emsa Sta�n�ng Solut�on and r�nse the culture d�shes or flasks w�th d�st�lled water to remove non-bound sta�n. R�nse unt�l water rema�ns clear.

7. D�scard the d�st�lled water �nto the b�ohazardous waste and allow the t�ssue culture d�shes or flasks to dry at room temperature.

3.2.2 Enumeration

CFU-F colon�es from human cells are typ�cally between 1 - 8 mm �n d�ameter and may be scored macroscop�cally. Photographs of representat�ve CFU-F-der�ved colon�es are shown �n Sect�on 3.3. Ensure that there �s a l�near relat�onsh�p between the cell numbers plated and the result�ng colony numbers, by confirm�ng that there are tw�ce as many colon�es when 2 x 106 cells are plated as compared to 1.0 x 106 cells. L�kew�se, there should be tw�ce as many colon�es when 1.0 x 106 cells are plated as compared to 0.5 x 106 cells. Ideally there should be 10 - 40 colon�es per 100 mm d�sh or T-25 cm2 flask. L�near�ty may not be observed outs�de of th�s range as the cells would have been under or overplated.

Each bone marrow sample �s un�que for that donor and the number of CFU-F may depend on a number of factors �nclud�ng age, presence of d�sease and prev�ous treatments g�ven to the pat�ent.

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3.3 Photographs of Human CFU-F-Derived Colonies

Med�um colony sta�ned w�th G�emsa Large colony sta�ned w�th G�emsa

Large colony sta�ned w�th G�emsa Large colony sta�ned w�th G�emsa

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4.0 Expansion of Cultured Mesenchymal Cells

Confluent mesenchymal cell cultures can be produced when cells from bone marrow are plated at relat�vely h�gh dens�t�es on t�ssue culture-treated flasks or d�shes �n Complete MesenCult® Med�um (Human). Mesenchymal cell numbers can then be expanded by passag�ng when the cells become 80% confluent �n the flask or d�sh. If cells rema�n �n a confluent state for a s�gn�ficant t�me (days) �t may reduce the�r longev�ty and the�r potent�al to d�fferent�ate. Culture-expanded mesenchymal cells can be used for a number of appl�cat�ons �nclud�ng plast�c�ty stud�es, assessment of d�fferent�at�on or expans�on potent�al, and the evaluat�on of phenotype.

4.1 Culture Set-up

1. Use the follow�ng table for recommended plat�ng concentrat�ons for processed human bone marrow cells. Plate the recommended cell number �n a T-25 cm2 flask �n 10 mL Complete MesenCult® Med�um (Human).

Cells must be plated using tissue culture-treated flasks.

4.2 Passaging Cultured Mesenchymal Cells

1. Check mesenchymal cells under a m�croscope to ensure that the cells are at an adequate stage for passag�ng (~80% confluence). Th�s should take approx�mately 7 - 10 days for pr�mary bone marrow cells but less t�me for culture-expanded cells. If the med�um �n the flask or d�sh appears ac�d�c (more yellow �n color than orange/red) pr�or to reach�ng 80% confluency, a half-med�um change can be done by remov�ng one half of the ac�d�c med�um and replac�ng �t w�th fresh Complete MesenCult® Med�um (Human) prewarmed to 37°C.

The proliferative ability of each marrow is donor-dependent and can be affected by a number of factors including age, disease or whether the sample comes from a transplant recipient. Therefore not all bone marrow samples may be confluent in a week and a half-medium change may help cells to proliferate in some samples.

2. Remove Complete MesenCult® Med�um (Human) from cultures. Adherent mesenchymal cells w�ll rema�n beh�nd. Wash the cells w�th PBS (Catalog #37350) to remove res�dual FBS-conta�n�ng med�um.

3. Add 5 mL Tryps�n-EDTA (Catalog #07901) to cover cells and �ncubate at 37°C for 3 - 7 m�nutes.

4. Check under m�croscope to ensure that the mesenchymal cells have detached. Add 1 mL FBS (Catalog #06471 or qual�ty cell culture tested equ�valent) to neutral�ze the act�on of tryps�n or alternat�vely add 5 mL of Complete MesenCult® Med�um (Human).

5. Collect tryps�n�zed cells �nto a 14 mL tube and centr�fuge the cells at 300 x g (~1200 rpm) for 8 m�nutes at room temperature w�th the brake on. Remove supernatant and resuspend pelleted cells �n Complete MesenCult® Med�um (Human).

6. The cells can now be d�v�ded �nto new t�ssue culture-treated flasks. The recommended d�lut�on �s 1/4 (e.g. one T-25 cm2 t�ssue culture-treated flask conta�n�ng 80% confluent mesenchymal cells can be passaged �nto four T-25 cm2 t�ssue culture-treated flasks).

Cell Source Cells Plated

Fresh BM treated w�th NH4Cl 1.0 - 1.5 x 107 mononuclear cells

Fresh BM pur�fied w�th F�coll-Paque™ PLUS 1.0 - 1.5 x 107 mononuclear cells

Fresh BM enr�ched us�ng the RosetteSep® Human Mesenchymal Stem Cell Enr�chment K�t

1.0 - 2.0 x 106 enr�ched cells

Frozen Marrow Stromal Cells (Catalog #MSC-001F) 1.25 - 2.5 x 105 cells

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4.3 Photographs of Cultured Mesenchymal Cells

Confluent mesenchymal cell culture (past opt�mal t�me for passag�ng).

Mesenchymal cell culture at ~40% confluency.

Mesenchymal cell culture at ~60% confluency. Mesenchymal cell culture at ~70% confluency.

Mesenchymal cell culture at ~80% confluency (�deal for passag�ng).

Mesenchymal cell culture at ~20% confluency.

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4.4 Phenotype of Cultured Cells

StemCell Technologies class�fies cultured human mesenchymal stem cells as be�ng negat�ve for the express�on of CD45 and CD34 and pos�t�ve for CD90 (THY-1), SH2 (CD105), and SH4 (CD73). The phenotype of cultured MSC �s therefore defined as: CD45-CD34-CD90(THY-1)+SH2(CD105)+SH4(CD73)+.

CD45- CD34- CD90+ (THY1) SH2+ (CD105) SH4+ (CD73)

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

CD45-PE CD34-PE CD90-PE SH2-FITC SH4-PE

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5.0 Helpful Hints and Frequently Asked Questions

5.1 Helpful Hints1. Thaw�ng of Mesenchymal Stem Cell St�mulatory Supplements should preferably be performed overn�ght under

refr�gerat�on (2 - 8°C). If th�s �s not poss�ble, thaw�ng of supplements �n a 37°C water bath �s perm�ss�ble.

Do not thaw supplement in a 56°C water bath.

2. Once prepared, the complete med�um �s stable at 2 - 8°C for one month. If the volume of the complete med�um exceeds your monthly requ�rements, �t �s poss�ble to al�quot the supplements and store at -20°C. Therefore, smaller volumes of complete med�um can be prepared ensur�ng the supplements represent one-tenth of the total volume (�.e. 10 mL of supplements to 90 mL of basal med�um). Do not freeze-thaw supplements more than tw�ce.

3. Cell counts should be performed �n 3% Acet�c Ac�d w�th Methylene Blue (Catalog #07060) to obta�n an accurate wh�te cell count.

4. T�ssue culture-treated d�shes must be used to support the growth of CFU-F and expans�on of cultured mesenchymal cells. StemCell Technologies recommends 100 mm culture d�shes (Catalog #27125/27127) or T-25 cm2 t�ssue culture flasks (Falcon Catalog #353109) or 6-well t�ssue culture treated plates (Falcon Catalog #353502 or Corn�ng Catalog #3506).

5.2 Frequently Asked Questions

1. Why don’t my cultured cells look like the photographs?

There are a number of potent�al reasons for abnormal morphology of cultured mesenchymal cells:

• Cells reached 100% confluency and were not tryps�n�zed �n a t�mely fash�on• Bone marrow sample was not fresh or stored properly• Bone marrow sample was from a pat�ent post transplantat�on (often not able to make confluent layer due to

low prol�ferat�ve potent�al)• Bone marrow sample was treated w�th spec�fic drugs such as 5FU

2. How long can I expect cultured mesenchymal cells to maintain their potential?

Usually culture-expanded mesenchymal cells can be ma�nta�ned for 6 - 8 passages depend�ng on the marrow. We have expanded cultures of mesenchymal cells for longer than 7 weeks and confirmed that phenotyp�cally the cells were no d�fferent at passage 8 than they were at passage 2.

Expansion of mesenchymal cells from 4 different bone marrow samples grown with Complete MesenCult® Medium (Human):

3. Can I grow or isolate mesenchymal stem cells from cord blood?

There are groups that have shown that MSCs can be �solated and cultured from cord blood samples when the cord blood �s very fresh (�.e. less than 6 hours old).32-34 We have not been successful when work�ng w�th samples that are processed and plated more than 6 hours from collect�on.

Bone Marrow Sample Fold Expansion Number of Passages Days in Culture

A 3730X 5 40

B 6970X 8 58

C 1770X 6 41

D 230X 4 28

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4. Can I cryopreserve cultured mesenchymal stem cells?

Mesenchymal stem cells can be frozen at any passage. Stud�es �n our laboratory have shown that cryopreserved cells from var�ous passage numbers 2 - 7 ma�nta�n the�r phenotype and d�fferent�at�on potent�al.

Recommended Protocol for Freezing Mesenchymal Cells

Before beg�nn�ng have all reagents COLD (2 - 8°C) and label ster�le cryov�als us�ng an �ndel�ble marker.

1. Make up 20% D�methyl Sulfox�de (DMSO) �n Fetal Bov�ne Serum (FBS; Catalog #06471 or qual�ty cell culture tested equ�valent) and filter ster�l�ze us�ng a 0.2 µm filter. Keep on �ce.

2. Harvest cells from the t�ssue culture surface (refer to Sect�on 4.2 for a suggested protocol). Centr�fuge cells and resuspend �n FBS to g�ve a max�mum concentrat�on of 2 x 106 cells per mL. Place th�s cell suspens�on on �ce.

3. M�x cells gently w�th 20% DMSO �n FBS at a rat�o of 1:1 (the final cell suspens�on w�ll be 90% FBS/10% DMSO). Transfer 1 mL of cells �n freez�ng med�um to each cryov�al. F�nal cell concentrat�on w�ll be ~1 x 106 cells per v�al.

4. Place cryov�als �mmed�ately �nto thawed 70% �sopropanol freez�ng conta�ner. Place conta�ner �n -135°C freezer overn�ght.

Do not let cells sit in freezing medium at room temperature. Keep on ice and transfer within 5 minutes to the freezing container.

5. On the next day, remove frozen v�als from the freez�ng conta�ner and store at -135°C or colder or �n l�qu�d n�trogen.

Recommended Procedure for Thawing Cells

1. Thaw cells qu�ckly �n a 37°C water bath or beaker of warm water �n a t�ssue culture hood. W�pe the cryov�al w�th 70% ethanol.

Do not vortex cells at any time.

2. Gently transfer cells �nto a 50 mL centr�fuge tube.

3. Slowly add 15 mL IMDM w�th 2% FBS (Catalog #07700) dropw�se wh�le hold�ng tube and gently sw�rl�ng.

4. F�ll tube to 50 mL w�th IMDM conta�n�ng 2% FBS. Gently �nvert tube to m�x.

5. Centr�fuge cells at 300 x g (~1200 rpm) for 8 m�nutes.

6. D�scard supernatant and ‘fl�ck’ tube gently to resuspend the pellet.

If cells are clumpy, add 0.25 - 0.5 mL of 1 mg/mL DNAse I (Catalog #07900) to the resuspended cells and repeat Steps 3 - 6.

7. Resuspend cells at des�red concentrat�on �n Complete MesenCult® Med�um (Human).

12







6.0 Mesenchymal Adipogenic Assay using MesenCult®

6.1 Culture Set-up

1. Thaw MesenCult® Ad�pogen�c St�mulatory Supplements (Catalog #05403) at room temperature or 2 - 8°C overn�ght.

2. Add the ent�re contents of the Ad�pogen�c Supplements to MesenCult® MSC Basal Med�um (Human; Catalog #05401) and m�x thoroughly. Th�s �s now referred to as Complete MesenCult® Ad�pogen�c Med�um. If less than 500 mL w�ll be requ�red �n one month, smaller volumes can be prepared. Al�quot supplements and prepare Complete Mesencult® Ad�pogen�c Med�um by us�ng the same method descr�bed �n Sect�on 3.1.1. Complete MesenCult® Ad�pogen�c Med�um should be stored at 2 - 8°C and used w�th�n one month.

3. Use the follow�ng table for recommended plat�ng concentrat�ons for processed human bone marrow cells. Plate the recommended cell numbers �n a T-25 cm2 flask �n 10 mL of Complete MesenCult® Ad�pogen�c Med�um.

4. Culture cells for 1 - 2 weeks. No med�um changes are requ�red unless med�um become yellow/orange �n color, �n wh�ch case a half-med�um change should be performed. Ad�pogen�c development w�ll be v�s�ble as fat globules �n cells �n spec�fic areas throughout the culture. Refer to Sect�on 6.2 for representat�ve photographs.

Cell Source Cells Plated

Fresh BM treated w�th NH4Cl 1.0 - 1.5 x 107 mononuclear cells

Fresh BM pur�fied w�th F�coll-Paque™ PLUS 1.0 - 1.5 x 107 mononuclear cells

Fresh BM enr�ched us�ng the RosetteSep® Human Mesenchymal Stem Cell Enr�chment K�t

1.0 x 106 enr�ched cells

Frozen Marrow Stromal Cells (Catalog #MSC-001F) 1.25 - 2.5 x 105 cells

13







6.2 Photographs of Human Adipocytes

Human ad�pocytes generated from confluent cultured mesenchymal cells.

Human ad�pocytes generated from confluent cultured mesenchymal cells.

Human ad�pocytes generated from ficolled bone marrow. Human ad�pocytes generated from ficolled bone marrow.

Human ad�pocytes generated from ficolled bone marrow, sta�ned w�th O�l Red-O.

Human ad�pocytes generated from ficolled bone marrow, sta�ned w�th O�l Red-O.

14







7.0 Mesenchymal Osteogenic Assay using MesenCult®

7.1 Preparation of Complete MesenCult® Osteogenic Medium

1. Fresh Complete MesenCult® Osteogen�c Med�um should be prepared weekly. The amount of med�um to be prepared should be based on the numbers of cultures that need med�um. Instruct�ons are prov�ded for the preparat�on of 50 mL of Complete MesenCult® Osteogen�c Med�um. To fac�l�tate th�s, reagents should be al�quoted and stored upon arr�val as descr�bed below:

• MesenCult® MSC Basal Medium (Human; Catalog #05401, 450 mL).O Al�quot �nto 10 x 45 mL and store at 2 - 8°C.

• Osteogenic Stimulatory Supplements (Human; Catalog #05405, 80 mL) used at 15% final volume.O Al�quot �nto 10 x 8 mL and store at -20°C.

• ß-Glycerophosphate (Catalog #05406, 10 mL, 1 M), used at a final concentrat�on of 3.5 mM �n human assays and 5.0 mM �n rat assays.

O Al�quot �nto 10 x 1 mL v�als and store at -20°C.

• Dexamethasone (Catalog #05407, 1 mg), used at a final concentrat�on of 10-8 M.O D�ssolve the powder �n a small volume of absolute ethanol and then add ethanol to a final volume of

25.5 mL to make a stock concentrat�on of 10-4 M.O Al�quot �nto mult�ple 500 µL v�als and store at -20°C.

• Ascorbic Acid (Catalog #07157, 100 mg), used at a final concentrat�on of 50 µg/mL. O D�ssolve the powder �n 10 mL of MesenCult® MSC Basal Med�um (Human) to obta�n a stock solut�on of

10 mg/mL. O Al�quot �nto 10 x 1 mL v�als and store at -20°C.

2. To prepare Complete MesenCult® Osteogen�c Med�um, p�pette 42.5 mL of MesenCult® MSC Basal Med�um (Human) �nto a 50 mL con�cal tube and add the follow�ng:

• 7.5 mL Osteogen�c St�mulatory Supplements• 5 µL Dexamethasone (10-4 M stock solut�on)• 250 µL Ascorb�c Ac�d (10 mg/mL stock solut�on)• 175 µL ß-Glycerophosphate (stock solut�on at 1.0 M) (if required, see Note below)

Note: ß-Glycerophosphate is not added to the complete medium at initiation of the assay. Typically ß-Glycerophosphate is added only after there is evidence, by phase microscopy, of cell multilayering.

Antibiotics and antimycotics may be added at the researcher’s discretion.

15







7.2 Culture Set-up

There are many protocols �n the l�terature that descr�be the development of osteogen�c cells from e�ther bone marrow or cultured mesenchymal cells5,35-38 wh�ch vary sl�ghtly �n the concentrat�ons of reagents used. The protocol below �s an example of a method that supports the growth of osteogen�c cells from human bone marrow.

Complete MesenCult® Osteogen�c Med�um also supports the prol�ferat�on of rat osteogen�c cells. The opt�mal concentrat�on of ß-Glycerophosphate �s 5 mM for rat cells.

1. Prepare cancellous bone fragments by m�nc�ng the bone �nto very small p�eces (1 - 3 mm �n s�ze).

2. Flush fragments w�th 20 - 30 mL of PBS (Catalog #37350) and then vortex the fragments w�th another 20 - 30 mL of PBS.

The fragments should appear almost white at this stage.

3. Pass the cell suspens�on through a 70 µm cell stra�ner (BD Catalog #352350) to remove bone fragments.

4. Centr�fuge cells at 400 x g (~1320 rpm) for 15 m�nutes.

5. D�scard the supernatant and resuspend cells �n PBS.

6. Place cells on F�coll-Paque™ PLUS and centr�fuge at 400 x g (~1320 rpm) for 25 m�nutes w�th the brake set to the “off” pos�t�on.

If unprocessed bone marrow cells are available, dilute the bone marrow 1/3 with PBS + 2% FBS and start at this step.

7. Remove the cells at the �nterface and resuspend the cells �n Complete MesenCult® Osteogen�c Med�um without ß-Glycerophosphate.

8. Seed cells �n t�ssue culture-treated flasks or plates at a concentrat�on of 1 - 2 x 105 cells per cm2.

9. Replen�sh the culture med�um after 5 days by remov�ng the med�um (and non-adherent cells). These cells and the med�um can be d�scarded. The cultures are replen�shed w�th fresh Complete MesenCult® Osteogen�c Med�um, aga�n w�thout ß-Glycerophosphate unless cell mult�layer�ng has been noted.

Multilayering is the layering of cells on top of each other, forming a matrix as opposed to growing in a planar manner. Multilayering is indicative of the beginning of bone generation.

10. Once mult�layer�ng has been observed, add ß-Glycerophosphate to Complete MesenCult® Osteogen�c Med�um as d�rected �n Sect�on 7.1. Cont�nue to replen�sh cultures w�th ß-glycerophosphate-conta�n�ng med�um every 2 - 3 days for a m�n�mum of three weeks (for rat cultures) or 5 weeks (for human cultures).

11. Osteogen�c cells may be detected by tetracycl�ne label�ng35 or von Kossa sta�n�ng. Cultures may be ma�nta�ned for extended t�me per�ods (>8 weeks) for other types of stud�es.


16







8.0 Appendix 1: RosetteSep® Human Mesenchymal Stem Cell Enrichment Cocktail

Complete �nformat�on concern�ng the enr�chment of mesenchymal stem cells from unprocessed human bone marrow us�ng RosetteSep® can be obta�ned at: http://www.stemcell.com/product_catalog/hmesenchymal.asp.

8.1 RosetteSep® Procedure (Catalog #15128/15168)

Ensure that bone marrow sample, PBS + 2% FBS (Catalog #07905), F�coll-Paque™ PLUS (Catalog #07957), EDTA, and centr�fuge are all at room temperature. Perform a nucleated cell count on fresh unprocessed bone marrow us�ng 3% Acet�c Ac�d w�th Methylene Blue (Catalog #07060). A 1/50 or 1/100 d�lut�on �s recommended.

1. Al�quot a known volume of bone marrow �nto a 14 mL tube. Add 50 µL RosetteSep® Human Mesenchymal Stem Cell Enr�chment Cockta�l per mL of bone marrow and m�x well.

2. Incubate 20 m�nutes at room temperature.

3. D�lute sample w�th tw�ce the volume of PBS + 2% FBS and 1 mM EDTA. M�x gently.

4. Layer the d�luted sample on top of the F�coll-Paque™ PLUS. Be careful to m�n�m�ze m�x�ng of F�coll-Paque™ PLUS and sample. See table below for volume recommendat�ons. W�th 50 mL centr�fuge tubes, we suggest us�ng a m�n�mum of 15 mL F�coll-Paque™ PLUS to make �t eas�er to remove the enr�ched cell layer.

Recommended Volume and Tube Sizes

Unprocessed Bone Marrow

PBS + 2% FBS + 1 mM EDTA

F�coll-Paque™ PLUS Tube S�ze

1 mL 2 mL 5 mL 14 mL

2 mL 4 mL 5 mL 14 mL

5 mL 10 mL 15 mL 50 mL

10 mL 20 mL 15 mL 50 mL

5. Centr�fuge for 25 m�nutes at 300 x g (~1200 rpm) at room temperature, w�th the brake off.

6. Remove the enr�ched cells from the F�coll-Paque™ PLUS : plasma �nterface.

Sometimes it is difficult to see the cells at the interface, especially when very rare cells are enriched. It is advisable to remove some of the Ficoll-Paque™ PLUS along with the enriched cells in order to ensure their complete recovery.

7. Wash enr�ched cells w�th PBS + 2% FBS and 1 mM EDTA.

8. We recommend that enr�ched samples are lysed w�th ammon�um chlor�de to remove res�dual red blood cells pr�or to flow cytometr�c analys�s (th�s can be done as one of the wash steps) or �f res�dual red blood cells w�ll �nterfere w�th subsequent assays. Resuspend cells �n Complete MesenCult® Med�um (Human) for culture or appropr�ate med�um for flow cytometr�c analys�s.


17







8.2 RosetteSep® Procedure Summary

Label

Unwanted cells are cross-linked to red blood cells (rosetted) with Tetrameric Antibody Complexes

Incubate 20 minutes at room temperature

Layer over Ficoll-Paque™ PLUS

Add RosetteSep® antibody cocktail

Ficoll-Paque™ PLUS

Spin

CollectPlasma

Enriched cells

Ficoll-Paque™ PLUS

Red cells and unwanted cells (rosetted)

Desired cells

Unwanted cells

Red cells

18







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344, 1977

2. Verfa�ll�e CM: Soluble factor(s) produced by human bone marrow stroma �ncrease cytok�ne-�nduced prol�ferat�on and maturat�on of pr�m�t�ve hematopo�et�c progen�tors wh�le prevent�ng the�r term�nal d�fferent�at�on. Blood 82: 2045-2053, 1993

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13. Galotto M, Ber�sso G, Delfino L, Podesta M, Ottagg�o L, Dallorso S, Dufour C, Ferrara GB, Abbondandolo A, D�n� G, Bac�galupo A, Cancedda R, Quarto R: Stromal damage as consequence of h�gh-dose chemo/rad�otherapy �n bone marrow transplant rec�p�ents. Exp Hematol 27: 1460-1466, 1999

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16. Qu�r�c� N, Sol�go D, Bossolasco P, Serv�da F, Lum�n� C, Del�l�ers GL: Isolat�on of bone marrow mesenchymal stem cells by ant�-nerve growth factor receptor ant�bod�es. Exp Hematol 30: 783-791, 2002

17 Deschaseaux F, G�ndraux F, Saad� R, Obert L, Chalmers D, Herve P: D�rect select�on of human bone marrow mesenchymal stem cells us�ng an ant�-CD49a ant�body reveals the�r CD45med,low phenotype. Br J Haematol 122: 506-517, 2003

18. Jones EA, K�nsey SE, Engl�sh A, Jones RA, Straszynsk� L, Mered�th DM, Markham AF, Jack A, Emery P, McGonagle D: Isolat�on and character�zat�on of bone marrow mult�potent�al mesenchymal progen�tor cells. Arthr�t�s Rheum 46: 3349-3360, 2002

19. Gronthos S, Zannett�no AC, Hay SJ, Sh� S, Graves SE, Kortes�d�s A, S�mmons PJ: Molecular and cellular character�sat�on of h�ghly pur�fied stromal stem cells der�ved from human bone marrow. J Cell Sc� 116:1827-1835, 2003

20 Joyner CJ, Bennett A, Tr�ffitt JT: Ident�ficat�on and enr�chment of human osteoprogen�tor cells by us�ng d�fferent�at�on stage-spec�fic monoclonal ant�bod�es. Bone 21:1-6, 1997

21. Clarke E, Wognum AW, Marc�n�ak R, Eaves AC: Mesenchymal cell precursors from human bone marrow have a phenotype that �s d�st�nct from cultured mesenchymal cells and are exclus�vely present �n a small subset of CD45lo, SH2+ cells. Blood 98: 355a, 2001

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22. Reyes M, Lund T, Lenv�k T, Agu�ar D, Kood�e L, Verfa�ll�e CM: Pur�ficat�on and ex vivo expans�on of postnatal human marrow mesodermal progen�tor cells. Blood 98: 2615-2625, 2001

23. J�ang Y, Jahag�rdar BN, Rhe�nhardt RL, Schwartz RE, Keene CD, Ort�z-Gonzalez XR, Reyes M, Lenv�k T, Lund T, Blackstad M, Du J, Aldr�ch S, L�sberg A, Low WC, Largaespada DA, Verfa�ll�e CM: Plur�potency of mesenchymal stem cells der�ved from adult marrow. Nature 418: 41-49, 2002

24. Lazarus HM, Haynesworth SE, Gerson SL, Rosenthal NS, Caplan AI: Ex vivo expans�on and subsequent �nfus�on of human bone marrow-der�ved stromal progen�tor cells (mesenchymal progen�tor cells): Impl�cat�ons for therapeut�c use. Bone Marrow Transplant 16: 557-564, 1995

25. Pere�ra RF, O’Hara MD, Laptev AV, Halford KW, Pollard MD, Class R, S�mon D, L�vezey K, Prockop DJ: Marrow stromal cells as a source of progen�tor cells for nonhematopo�et�c t�ssues �n transgen�c m�ce w�th a phenotype of osteogenes�s �mperfecta. Proc Natl Acad Sc� (USA) 95: 1142-1147, 1998

26. Horw�tz EM, Prockop DJ, Mar�n� JC, F�tzpatr�ck LA, Gordon P, Koo W, Neel M, Orchard P, Brenner M: Bone marrow transplantat�on to correct the mesenchymal defect of ch�ldren w�th osteogenes�s �mperfecta. Blood 92: 249, 1998

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