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Background - Cellular Therapies
 
  • A Quick Guide for Parents,

    Patients & Non-Scientists

  • Stem cell "Quality" Testing

  • Stem Cell Potency Testing

Background to Cellular Therapeutics

 

"The Power of Stem Cells": A Quick Guide for Parents, Patients and Non-Scientists

 

Introduction

Blood stem cell transplantation dates back more than 60 years. It was not until the 1970's that transplantation of human bone marrow stem cells became a routine procedure to cure malignancies of the blood system. In the 1980s, investigators learnt how to mobilized stem cells out of the bone marrow and into the circulation. This led to the use of mobilized peripheral blood as a stem cell source for transplantation. In 1988, umbilical cord blood ceased to be a waste product and became the third source of hematopoietic stem cells used for transplantation into patients with blood malignancies.

Since that time, collecting and storing umbilical cord blood has become big business. Cord blood banks can now be divided into private, public and hybrid banks. Private cord blood banking has become a very competitive business all vying for the opportunity to entice parents to bank their baby's cord blood. Although, private cord blood banks usually have to register with a regulatory agency, they do not function under the same regulatory requirements as a public cord blood bank. Public cord blood banks rely on the donation of cord blood for which they usually receive a subsidy from the government. These banks are under regulatory scrutiny. Hybrid banks are a mixture between a private and public bank.

At one time, bone marrow transplantation centers used fresh bone marrow, provided by the donor, processed and administered to the patient on the same day. Nowadays, like umbilical cord blood and mobilized peripheral blood, bone marrow cells are processed and then cryopreserved and stored in liquid nitrogen before being used in a patient.

 

Why Are Stem Cells So Important?

Without the stem cells, there would be no stem cell transplantation procedure. However, the "Power of the Stem Cells" is based on 3 properties that no other cells demonstrate:

  1. Stem cells have the capacity to self-renew. This means that when stem cells are transplanted into a patient, they must be able to build up their own numbers in order for the patient to reconstitute their new blood-forming system.
  2. Stem cells have the capability of producing red blood cells, platelets and white blood cells, including granulocytes and cells of the immune system.
  3. These two stem cell properties are absolutely dependent upon the ability of the transplanted stem cells to proliferate and grow to produce more cells. Without stem cell proliferation, no stem cell engraftment and reconstitution would occur.

 

The "Power of Stem Cells" is Based on Their Health

For cord blood, bone marrow or mobilized peripheral blood stem cells to be used safely and effectively, the stem cells must be healthy. This means that the stem cell must demonstrate their ability to proliferate and grow. However, stem cell proliferation must be above a certain level. This is called the "acceptance level". If the stem cells do not demonstrate proliferation above this level, there is a high chance, that when used, they will not be able to proliferate properly, if at all, and the transplantation procedure will be a failure.

Therefore, the health of the stem cells should be a Number 1 Priority, not only for all cord blood banks, but also for stem cell transplantation center that carry out the procedure. Unfortunately, this is not the case.

The stem cells in fresh umbilical cord blood, bone marrow or mobilized peripheral blood demonstrate about a 3-4 fold ability to proliferate above that for frozen cells. This reduced stem cell health has very important implications.

It means that extra attention must be paid to the stem cells to ensure that they are useful for the important transplantation procedure. Although cord blood stem cells have been stored and used after many years in liquid nitrogen, no systematic study has every been performed to determine whether stem cell health deteriorates with time in storage.

To date, more than 800,000 cord blood units have been donated to public cord blood banks in the U.S.A. About 40,000 cord blood transplants have been performed worldwide. Millions of cord blood units have now been stored in private cord blood banks. However, not a single cord blood unit has ever been tested to ensure that the stem cells are healthy.

 

The Reasons Why Stem Cell Health is Not Tested

Stem cell transplantation is dependent upon stem cell health. If stem cells are not healthy, graft failure can occur. The tests used by stem cell transplantation centers and public cord blood banks do not test for stem cell health. The tests currently used, and which have not changed for more than 25 years, are based on total cell count, the viability of the cells (whether the cells are alive or dead) and the presence of a marker on the surface of stem cells and other cells called CD34. None of these determine stem cell health. Another test is used to try and grow cells in culture so that they form colonies. The colonies are counted and this is supposed to give an indication of whether the cells will grow. However, not only is this test insensitive to detect the very small number of stem cells available, but the variability of the results is so high that no definitive information can be obtained.

Private cord blood banks are not required to perform any of these tests, although the total cell count and viability will usually be performed routinely. Occasionally, and at random, samples will be tested for CD34 and the ability of the cells (not necessarily the stem cells) to grow.

 

Parents and Patients Take Note

Private cord blood banks worldwide invest a lot of money into the "Power of Stem Cells" so that parents save the cord blood and/or cord tissue. However, parents should be aware that the money they spend for collecting, processing and storing the cord blood cells does not include any testing to ensure that the stem cells are healthy. Parents should also be aware that during the time the cord blood cells are stored, testing is never performed to ensure that the stem cells remain healthy.

Public cord blood banks and stem cell transplantation centers do not perform any tests to ensure that the stem cells are healthy prior to use.

 

How HemoGenix® Measures Stem Cell Health

HemoGenix® was the very first company to develop tests to measure stem cell health. This started in 2002 and since that time, the company has continued to advance the technology. In 2005, it developed the HALO® SPC-QC test, which is used to measure stem cell health in very small amounts of cord blood, bone marrow or mobilized peripheral blood. This test makes use of the fact that if cells can proliferate and grow, they will increase their chemical energy. This chemical energy is contained in a molecule called ATP (adenosine triphosphate). All cells make ATP as their chemical energy source. After culturing the cells for 5 days, the chemical energy is measured using a method similar to that used by fire flies to produce light. It is the amount of light that indicates the health of the stem cells. Only HemoGenix® has this highly advanced stem cell technology.  

 

Why HemoGenix® Cares and How We Can Help

HemoGenix® is the leader in testing stem cell health and other properties of stem cells. Using our advanced stem cell technology, we have been able to prove that many assumptions and aspects of cell processing and testing, especially for umbilical cord blood, that have been used for years, are scientifically incorrect. We believe that parents and patients are entitled to the highest quality stem cell product. However, present testing does not allow this to happen. There is room for significant improvment to reduce risk and improve safety and efficacy. That is why we have advocated for many years that testing for stem cell health is of primary importance.    

There are many reasons why cord blood banks and stem cell transplantation centers do not offer these tests. The primary reasons are that they are afraid of any change and that new technology might highlight the imperfections of current testing. In fact, the information obtained using tests from HemoGenix® far outweigh any reason for not measuring stem cell health.

It is therefore up to parents and patients to understand that the minimal testing performed by cord blood banks and stem cell transplantation centers does not include the most important testing of stem cell health.

Parents and patients should either contact the cord blood bank or transplantation center to request that stem cell health is measured. Alternatively, contact HemoGenix® directly and we will contact the cord blood bank or stem cell transplantation center on your behalf to ensure that your request is fulfilled.

Stem Cell "Quality" for Engraftment and Reconstitution

 

Download the article "Detecting primitive hematopoietic stem cells in total nucleated and mononuclear cell fractions from umbilical cord blood segments and units" published in the Journal of Translational Medicine (2015) 13:94. Click here to download or go to PubMed. This article used HALO®-96 SPC-QC.

 

What is Stem Cell "Quality"?

Stem cell "quality" is the proliferation ability of the stem cells measured at a specific cell dose and point in time. Since one of the properties of stem cells is to proliferate, stem cell "quality" can only be measured using a stem cell proliferation assay. Stem cell "quality" is therefore a specific biological function that characterizes the stem cell cells. 

Regulatory Considerations

The Stem Cell Therapeutic and Research Act of 2005 requires that all public cord blood banks ensure that the cord blood units collected and maintained are "high-quality cord blood units". Present tests, notably:

  • Total nucleated cells (TNC),
  • Viability, and
  • Viable CD34+ cells, together with the
  • CFU assay

are considered "Minimal Testing Criteria" and do not determine if a cord blood unit is actually of "high-quality". The assays described below promote "Best Practice Criteria Testing" and have been designed to measure stem cell "quality". 

Assays for Stem Cell "Quality" 

HemoGenix® has developed 2 distinct assays to measure stem cell "quality".

 

 

Using STEMpredict to predict cord blood quality, viability and functionality

 

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STEMpredict™

STEMpredict™ is part of the HALO® family of assays for hematopoietic stem cell therapy. STEMpredict™ is the most rapid stem cell assay available. It is completed in just 3 days. As its name implies, STEMpredict™ predicts the ability of hematopoietic stem cells to demonstrate proliferation. It uses ATP bioluminescence technology to compare the stimulation and growth of stem cells to a background control (see the graph). STEMpredict™ is used to help determine whether a cord blood unit can be permanently banked and if a donor or patient has been properly mobilized for peripheral blood stem cell collection. 


HALO®-96 SPC-QC

HALO®-96 SPC-QC is a 5 - 7 day assay used to measure stem cell proliferation ability ("quality") before and after a processing procedure in order to standardize and optimize the method. HALO®-96 SPC-QC assay kits are available to measure either primitive hematopoietic stem cells (CFC-GEMM) alone or in combination with primitive lympho-hematopoietic stem cells (HPP-SP). Like STEMpredict™, it incorporates ATP bioluminescence technology required to detect and measure rare, stem cell populations.

For the mesenchymal stem cell system, the equivalent assay to HALO®-96 SPC-QC is MSCGlo™-96 HuQC

Assays for Engraftment and Reconstitution

After infusion of a hematopoietic stem cell product into a patient (the transplantation procedure), the stem cells will try to "home" or find their way to the bone marrow. There, they will lodge and start to proliferate. This is called the engraftment process. Shortly after early engraftment, some of the stem cells will enter the differentiation lineages that are responsible for producing red blood cells, neutrophils and platelets. This process is called reconstitution. A patient is said to have engrafted if the number of neutrophils is equal to or greater than 500,000/µL for 3 days or more and the number of platelets is equal to or greater than  20,000 - 50,000/µL for 3 days or more. The time at which these numbers are reached is call the Time to Engraftment. 

The cells from which the neutrophils are derived are the granulocyte-macrophage progenitor cells. Similarly, platelets are derived from megakaryocyte progenitor cells and red blood cells are derived from erythropoietic progenitor cells. The assay used to determine these progenitor cells is called the colony-forming unit (CFU) or colony-forming cell (CFC) assay. Since the assay takes 14 days until results are obtained and the time to engraftment for bone marrow or mobilized peripheral blood is similar to or less than this time period, the CFU/CFC assay is not a routinely used by many transplantation centers for these tissues. Umbilical cord blood, however, is always used from a cryopreserved source. It takes much longer for thawed cord blood cells to start reconstituting a patient (usually 20 days or longer) and as a result, the CFU/CFC assay has been maintained as a requirement by the cord blood community.

Usually, the CFU/CFC assay is performed prior to cord blood unit cryopreservation and does not provide a good indicator for thawed cells. Indeed, the assay is not only difficult to perform, but exhibits notoriously high variations (coefficients of variation, CVs) that make inter-laboratory comparisons extremely difficult. In their Current Inventory Requirements for New Cord Blood Units, the NMDP only requires a sample tested using the CFU/CFC assay to be reported as "Growth" or "No Growth" with the actual colony count. That the threshold for cord blood unit release cannot be defined has led to the conclusion that the assay should not be used or a different assay should be used.  

 

HALO®-96 PMT

HALO®-96 PMT is an alternative the CFU/CFC assay. It is another member of the cellular therapy assay family designed to predict time to engraftment and determine "global" reconstitution. Early prediction of engraftment can provide much needed time for the transplantation physician to re-treat the patient if engraftment is not predicted engraftment or is delayed. Therefore, the greater the predictive value of the assay, the lower the risk to the patient.

 

Patient monitoring after a stem cell transplant using HALO-96 PMT 7-Population "Global" assay

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In addition, HALO®-96 PMT for 4-, 5- or 7-populations can be used to monitor hematopoietic or lympho-hematopoietic reconstitution. The graph shows results of a 7-population assay for both bone marrow and cord blood detected after 7 days in culture. If results are obtained after only 5 days, the response of cord blood will be much lower than that of bone marrow. This indicates that cryopreserved cord blood cells require about 2 days longer to reach the same level of proliferation ability than bone marrow cells.

Stem Cell Potency Testing

  

Promoting "Best Practice Criteria Testing" for Hematopoietic Stem Cell Therapy products

 

 

 

Published articles on measuring hematopoietic stem cell potency

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Regulatory Considerations

The potency of a drug predicts its dose. Traditional drug potency testing has been a routine procedure for many years. Potency testing of cellular therapeutic and regenerative medicine products is a new and often misunderstood and confusing experiance. Since regulatory agencies, i.e. FDA and the European Medicines Agency (EMA), are new to this field, they rely on industry to provide them with details. This is particularly the case for stem cell biologics and hematopoietic stem cell therapy in particular.

The EMA published its guidelines on potency testing in May 2008 entitled "Guideline on the Potency Testing of Cell Based Immunotherapy Medicinal Products for the Treatment of Cancer", while the FDA published its "Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products" in January 2011. The tissue most affected by these guidelines at the present time is umbilical cord blood since this was designated as a "drug" by the FDA in 2009. As such, the potency of cord blood stem cell products that are minimally manipulated and used for allogeneic transplantation should be assessed when a unit has been identified and prior to use in the patient. This is because "All potency assays used for release testing of licensed drug products must comply with applicable biologics and cGMP regulations including":

 

Potency Assay Requirements Code of Federal Regulations
Indicates biological activity specific to the product 600.3;610.3;210.3(b)(16)(ii)
Results allow for realease of the product 610.1; 211.165(a)
Provides quantitative data 211.194
Meets pre-defined acceptance/rejection criteria 211.165(b)(16)(ii); 211.160
Includes reference material, standards and controls 210.3(b)(16)(ii); 211.160
Demonstrates validation 211.165(e); 211.194(a)(2)
Measures identiy & activity of the active ingredients 211.164(a)

 

In contrast to these regulations, non-binding recommendations by the FDA for purity and potency of cord blood represent "Minimum Testing Criteria" and include: 

  • Total nucleated cells (TNC)
  • Viable nucleated cells
  • Viable CD34+ cells
  • All tested on the pre-cryopreservation product.

These same recommendations are mirrored in the NetCord-FACT 5th Edition Standards, but in addition include the statement, "CFU or other validated potency assay". This assumes that the colony-forming unit or CFU is a potency assay and is validated, neither of which are correct.

Since the potency and general characterization of umbilical cord blood stem cells has never been addressed by either the FDA or standards organizations (NetCord-FACT, AABB, NMDP), cord blood testing has not been in compliance with the Stem Cell Therapeutic and Research Act of 2005.

 

How does "Minimum Testing Criteria" differ from "Best Practice Criteria Testing"?

The 'minimum testing criteria" refers not only to the most basic type of testing that can be performed, but also to tests and assays that are not standardized or validated and, as a result, produce highly variable data that cannot be used to produce statistical significance. "Best practice criteria testing" are often more complex assays that allow the method to be standardized and validated so that consistency, reliability and reproducibility can be obtained. Implementing "best practice criteria testing" includes, but is not limited to, the following considerations: 

  • Intsrument-based signal detection readout
  • Quantitative data
  • Uses standardized analysis of biological assay procedures described in the U.S. Pharmocopeia
  • Inclusion of internal and/or external standards that allow a standard curve to be performed
  • Inclusion of controls
  • Determination of acceptance/rejection values
  • Determination of value ranges for specific assay parameters
  • Potential to validate the assay (accuracy, sensitivity, selectivity, reliability, reproducibility, robustness)
  • Ability to accurately and reliably measure the "active" target components
  • Ability to compare data over time. 

The key to "best practice criteria testing" is standardized measurement. If an assay is not standardized, it cannot be validated and the results cannot be trusted.

 

What is Stem Cell Potency?

Stem cell potency is the quantitative and validated measurement of the biological activity of the "active" stem cell components that are responsible for the intended response or effect.

From the regulatory requirements for a potency assay, it is clear that "minimal criteria testing" cannot and does not provide the compliance needed to measure potency of a hematopoietic stem cell therapeutic product. To measure stem cell potency, the assay has to conform to "best practice criteria".

 

 

When should stem cell potency be measured?

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When should cord blood stem cell potency be measured?

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When Should Potency be Measured?

The timing of conducting a potency assay is key to providing the transplant center and physician with the most important information. When an umbilical cord blood unit is collected, it must be cryopreserved within 48 hours of collection. When the unit reaches the processing laboratory a sample is taken on which numerous tests and assays are performed, including HLA typing, sterility, infectious diseases as well TNC, viability, CD34 and CFU, i.e. "minimal testing criteria". It can take up to three weeks to obtain all the results from a single, by which time the unit has been frozen. "Minimal testing criteria" are usually performed on the fresh cells and the decision to upload the umbilical cord blood unit to the NMDP cord blood inventory is based on parameters such as cell number, volume, weight etc., rather than the properties of the stem cells present in the product. Prior to freezing, the cord blood unit is red cell reduced. However, the properties of the cells are quite different between the fresh and frozen state. In fact, stem cell proliferation ability will actually decrease 3-4 fold between the fresh and frozen state. It therefore follows that the "minimum testing criteria" recommended by the FDA as purity and potency characteristics on a pre-cryopreserved sample cannot be used to measure cord blood stem cell potency. Indeed, due to these testing method not complying with the Code of Federal Regulations for potency assays as well as other considerations, the "minimum testing criteria" should never be considered as potency assays.

For umbilical cord blood, stem cell potency must be performed on a sample after the unit has been cryopreserved (see diagram). This allows the results from the potency assay to be uploaded to the cord blood inventory database so that the transplant physician can asses whether the units with exhibit "high-quality" and high potency to predict engraftment. Since cord blood units may remain frozen for more than 15 years, a second potency assay should be performed when the unit has been identified as compatible with a patient and prior to leaving the cord blood bank for the transplant center. This allows results to be compared with the original potency assay to determine if stem cell "quality" and potency have changed and provides the transplant physician with the assurance that the unit can be used. 

 

Potency Assays that Conform to "Best Practice Criteria Testing"

HemoGenix® has developed two potency, quality, release assays that are compliant with both potency assay regulations and "best practice criteria testing". These are HALO®-Potency for hematopoietic stem cell products, MSCGlo™-Potency for mesenchymal stem cell products and STEMGlo™-Potency for ES, iPS and primary stem cell populations. Both incorporate ATP bioluminescence technology and like all HemoGenix® ATP bioluminescence assays, they incorporate standards and controls that promote "best practice criteria testing". In addition, they also allow an internal proficiency test to be performed evry time prior to measuring a sample. HemoGenix® has a Master File for HALO®-Potency with the FDA.

To perform a potency assay for a hematopoietic stem cell product (cord blood, mobilized peripheral blood, bone marrow or purified populations from these tissues), it is first necessary to reduce or remove unwanted cells (red blood cells, granulocytes, platelets) since these dilute and mask the rare stem cell populations that need to be determined.

HALO®-Potency, MSCGlo™-Potency and STEMGlo™-Potency are all performed by measuring the slope of the cell dose response of the stem cells being measured. The slope is a measure of stem cell proliferation potential. The steeper the cell dose response, the greater the stem cell proliferation potential, the more primitive the stem cells and the greater their potency. As seen in the diagram below, stem cell self-renewal, primitiveness and potency all correlate with stem cell proliferation potential. The greater the potency, the greater the probability of engraftment. Thus, a stem cell potency assay predicts the ability of the stem cell to engraft in the patient. This is called stem cell engraftment potential.

 

 

The hierarchy and organization of the blood-forming system and the properties and tests used to measure different cells

 

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The stem cell transplantation process and why stem cell potency must be measured before transplantation

 

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Stem Cell Potency / Engraftment Potential is not the same as Time to Engraftment

Time to engraftment is measured by the number of neutrophils, platelets and red blood cells in the circulation. These functional, mature cells are derived from lineage-specific progenitor cells, which, in turn, are derived from the hematopoietic stem cells. The number of these progenitors (GM-CFC, Mk-CFC and BFU-E) is indicative of time to engraftment. Since the presence of these progenitor cell populations and their functionally mature progeny are part of the reconstitution program as a result of engraftment, these populations provide no indication of stem cell engraftment potential and therefore cannot be used to measure the potency of the stem cell populations responsible for engraftment. The difference beteen engraftment potential and time to engraftment is shown in the lower diagram.