Prepacyte Based Bone Marrow Preparation United Stem Cell Technologies

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Prepacyte Based Bone Marrow Preparation United Stem Cell Technologies Powered By Docstoc
					          Isolation of mesenchymal stem cells
          expressing BMPs from vertebra and
              iliac crest during spinal fusion


  Tung T. Nguyen, MD+ ; Gary D. Fleischer#, MD (Spine and Brain Center of New England,
                         Nashua NH); Dan Collins, PhD* (CMDG, Minneapolis, MN)


+Consultant Stryker Spine, Trans1 Corp, shareholder USCT ; #Consultant Stryker Spine, Zyga technologies, Trans1 Corp; SAB Trans1, shareholder
                                                          USCT ;* principle CMDG
                 Purpose:

• To evaluate quantity and osteogenic potential
  of mesenchymal stem cells isolated from
  bone marrow of cervical and lumbar vertebra
  and iliac crest by PrepaCyte-MSC cell-
  separation medium.

                                                2
        Background Context
• Mesenchymal stem cells (MSC) are present in
  bone marrow, and are the osteoprogenitor cells
  responsible for bone fusion. Previous studies
  have identified progenitor cells with osteogenic
  potential in vertebra and iliac crest, but
  expression of BMP-2 and BMP-7 by these cells
  has not been described.

                                                     3
  Methods for Processing BMA
• Centrifugation
    –   SpineSmith
    –   Sepax (BioSafe)
                                       • GOALS
    –   Thermogenesis
    –   Res-Q 60 BMC
                                         – Cheaper
• Non Centrifuge based
    – LSMs
                                         – Easier
    – Density gradients
• Fat derived stem cells
                                         – Safer
    – No osteoprogenitor cells
    – No HSCs
                                         – Better Cells
    – Different intercellular milieu



                                                          4
                 AABB 2011 Meeting
                   Better Quality
• INSTITUTIONS (ALL): 1. St. Louis Cord Blood Bank and
  Cellular Therapy Laboratory, SSM Cardinal Glennon
  Children's Medical Center, St. Louis, MO, United States.
• Results/Findings: To date, 33 PrepaCyte-CB® CBUs have
  been shipped by the SLCBB for use in human transplantation.
  Outcomes data is available for 9 (27.3%) of these transplants.
  For the units that can be evaluated, all patients (n=9) have
  achieved neutrophil recovery with a mean time to recovery of
  14.2 days. This is an impressive mark for cord blood and is
  significantly lower than the bank’s historical experience of
  22.4 days (P<0.01). Additionally, there have been no infusion
  related adverse events reported for the patient population.

                           USCT - Confidential                 5
Prepacyte Based BMA Processing


• Processing procedure
  – BMA mixed in sterile mixing vessel
  – Gravity settling
     • Prepacyte utilizes surface chemistry of the undesired cells
     • Causes aggregation and rouleau formation
  – Supernatant contains desired cells
  – Crisp interface layer



                             USCT - Confidential                     6
                  Design of Study
• Double –Blind 50 patient study of bone marrow utilized in spinal
  fusion procedure. Five ml of sample from marrow used in procedure
  was used for the in vitro study.
• Marrow is processed by PrepaCyte-MSC to isolate MSC/Osteogenic
  Progenitor Cells
• MSC are enumerated in original sample and after isolation to
  determine the stem cell content of the original sample and determine
  the recovery of those cells. Cells are expanded in culture to determine
  expansion capacity and maturity of cells
• MSC are analyzed for expansion rate, osteogenic potential and
  production of BMP-2 , BMP-7, mineralization and expression of
  osteocalcin
• Data will be compared to patient outcome (rate of fusion) to
  determine clinical significance
              Obtaining Specimen

• IRB-approved protocol
• 50 patients undergoing anterior cervical fusion and posterior lumbar
  fusion
• Bone marrow aspirated from exposed vertebra/pedicle (N=30), or
  iliac crest (N=20)
• Specimens shipped to outside reference lab for processing
    – Flow cytometry for cell count and surface marker expression
    – Expansion of MSC in culture
    – Characterization of expanded MSCs -
  Depletion of Specific Cell Populations with
    Different Formulations of PrepaCyte

Monocytes




                    Granulocytes
      Lymphocytes



            Original Sample           Processed with        Processed with
                                       PrepaCyte-CB         PrepaCyte-MSC
                                   Only the erythrocytes     Erythrocytes,
                                       are removed         granulocytes and
                                                            monocytes are
                                                               removed
Isolation of MSC Osteogenic Progenitors


 1. Human bone marrow is mixed 5:7
    with PrepaCyte-MSC reagent


 2. Sample is mixed by inversion for
    30 minutes on a platform rocker

 3. Sample is placed upright for 30
    minutes. Lymphocytes and
    progenitor cells are collected in
    supernatant
          Isolation of Osteogenic
                 Progenitors

4.   Cells from the supernatant are
     cultured in a cell culture flask
     overnight and the MSC will
     adhere to the plastic.
     Lymphocytes and other cells
     are washed away and can be
     recovered, if desired.
                                Results
                                  > 106        105 to 106     104 to 105     > 104

                                   N=10            N=6            N=1
Cell Number       Iliac crest    mean 1.48e6    mean 6.04e5    mean 7.05e4
from Culture                       N=13           N=14            N=1          N=5
                  Vertebrae      mean 1.38e6    mean 6.47e5    mean 6.0e4       0

CD34 in           iliac crest     1.77E+06       9.05E+05       1.02E+06
original sample
                  Vertebrae       1.04E+06       4.32E+05       5.38E+05     1.83E+05

CD90/105 in       Iliac crest     1.79E+05        1.15E+04      2.80E+04
original sample
                  Vertebrae       1.72E+05       2.84E+04       4.34E+05     1.20E+04
Development of MSCs in Culture
      1-3 days in   Spreading
      culture




     Logarithmic    Confluence
     growth
Culture-expanded MSC



        Tubulin/Actin
BMP-2                   BMP-7
Differentiation to Osteoblast



      BMP-2            BMP-7




    Osteocalcin       Alizarin Red
                    Results
BMP-2 and BMP-7

-Present in 16/17 (94%) iliac crest samples
-Present in 27/33 (82%) vertebral donation sites (cervical vertebra, lumbar
pedicles)


Multivariate analysis of these factors showed NO impact on ability to isolate
MSCs in culture

- Sex of the patient –
- Volume size of sample –
- Current smoking history -
                                  Significance
•   Previous studies have shown that significant losses of osteogenic progenitors are associated
    the current methodologies to isolate osteogenic progenitors
•   PrepaCyte –MSC resulted in recoveries of 87.3% for CD34+ cells (HSC) and 92.8%
    recovery of CD90/105+ cells (MSC/osteogenic progenitors)
•   Processing by PrepaCyte-MSC resulted in significant removal of pro-inflammatory cells,
    81% removal of Granulocytes and 63% removal of Monocytes.
•   Donation location may contribute to harvest of Osteogenic Progenitors. Iliac crest samples
    resulted in 17/17 positive cultures, while spinal donations resulted in 28/33 positive
    cultures.
•   Volume of Sample, sex, smoking status was not associated with culture results. CD34+ and
    CD90/105+ cell numbers were.
•   Not all bone marrow samples are equal, quantitative metrics will aid in the development of
    standards for therapeutic dosage.
Thank You

				
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posted:10/3/2012
language:English
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