HemoGenix, Inc

Bone Marrow Toxicity/Stem Cell Hematotoxicity Testing

All studies are customized to the client's goals.

Bone marrow toxicity, also called myelotoxicity, occurs when a drug or other agent causes a decrease in blood-forming cells that result in anemia, neutropenia, thrombocytopenia or lymphopenia. Stem cell hematotoxicity occurs when the common stem cells that produce all of the blood-forming lineages are affected. In general, the stem cells are more sensitive than any other cells. Damage to the stem cells can result in the obliteration of the whole blood-forming system. Difference in bone marrow toxicity and stem cell hematotoxicity can depend on the species being examined. HemoGenix has developed patented and proprietary testing platforms to study and predict toxicity to the blood-forming system. HALO®-Tox HT is the most accurate and sophisticated stem and progenitor cell hematotoxicity assay platform developed with high throughput screening capability.

Applications

• High throughput predictive in vitro hemotoxicity screening
• Drug optimization
• Predictive in vitro hemotoxicity testing
• Ex vivo hemotoxicity testing (performed in collaboration with a CRO)
• Risk and safety assessment
• Drug-drug interaction and drug combination studies
• Mechanism of action studies
• Stem cell quality and potency assessment of cellular therapeutic products
• General studies on the blood-forming system

Cell Types

• Primary stem cells (up to 7 stem cell populations)
• Progenitor cells (up to 9 populations)
• Precursor cells (up to 5 populations)

Proliferation / Cytotoxicity Assays

• ATP bioluminescence assays (HALO-Tox HT for high throughput screening and testing. 96- or 384-well plate standardized and validated assays.)
• Fluorescence assays (HemoFLUOR-96)
  -Absorbance assays (HemoLIGHT-96)

Differentiation / Cytotoxicity Assays

• Colony-forming unit or cell (CFU/CFC) assays (ColonyGro and CAMEO-4)

Proliferation and Differentiation / Cytotoxicity Assays

• Colony-forming unit or cell (CFU/CFC) assay combined with a standardized and validated ATP bioluminescence readout (CAMEO-96)

Mechanism of Action (MOA) Studies
Most of the above assays can be multiplexed with the following assays and procedures:

• Genotoxicity
• Dye exclusion viability
• Mitochondrial dysfunction and metabolic viability
• Apoptosis
• Flow cytometry (cell cycle, polidy, membrane expression analysis)
• Oxidative stress and oxidative DNA damage
• Growth factor production / release

HemoGenix is the only company that has developed assays specifically for stem cell quality and potency testing of cellular therapeutic and regenerative medicine products.

At the present time, these assays are specifically for hematopoietic stem cell products for transplantation and mesenchymal stem/stromal cell products used for regenerative medicine.

HemoGenix stem cell potency assays have been validated according to FDA Bioanalytical Method Validation Guidelines and are compliant with FDA potency assay regulations.

Hematopoietic stem cell potency assays are usually performed on cryopreserved cells and take 7 days to complete with results available 1 day later. MSC potency assays are also performed on cryopreserved cells, which usually need to be passaged prior to quality and potency measurement. The assay is usually completed in 3-5 days and results are available 1 day later. In most cases, a Certificate of Potency is provide with the results.

All studies are customized to the client's goals.

The immune system is often considered a separate entity, but mature T- and B-lymphocytes are derived from primitive lymph-hematopoietic stem cells. As such immunotoxicity can be considered a a 2-tier system, the first tier being played out in the bone marrow, while the second tier is played out in the lymph tissues of the body and the circulation.

Tier 1: High Throughput In Vitro Immunotoxicity Testing on Bone Marrow
HALO-Tox HT (also used in hemotoxicity testing), a standardized and validated ATP bioluminescence assay is used to determine whether compounds are potentially cytotoxic on:
Very primitive lymph-hematopoietic stem cells
T-lymphocyte progenitor cells (T-CFC)
B-lymphocyte progenitor cells (B-CFC)

CAMEO-4 and CAMEO-96 can be used on the same cell populations to study effects on the differentiation process.

Tier 2: In Vitro Immunotoxicity Testing to the Immune System
ImmunoGlo-Tox HT, another proprietary standardized and validated ATP bioluminescence assays specifically designed for immune cell function is used and often multiplexed with flow cytometry to study innate and aquired immunity. These studies often involve the use of peripheral blood mononuclear cells and immune subpopulations such as CD4, CD8, dendritic cells and NK.

• T-lymphocyte proliferation
• B-lymphocyte proliferation
• Mixed lymphocyte reaction (using ImmunoGlo-MLC)
• Cytotoxic T-cell (CTL) response
• NK Response
• Mitogen stimulation
• Dendritic cell maturation and co-stimulation
• Cytokine production and release
• Immunostaining of CD4/CD8 cells and subpopulations
• Immunostaining of NK cells
• Immunostaining of B-cells
• Phagocytic ability
• Cell migration

Mechanism of Action (MOA) Studies

• Cell cycle
• Intracellular and membrane expression
• Mitochondrial dysfunction
• Oxidative stress
• Oxidative DNA damage
• Apoptosis
• Growth factor, interleukin, chemokine production and release

Toxicity to the Mesenchymal Stem Cell (MSC) system has far-reaching effects. As the cells responsible for adipogenesis, chondrogenesis and osteogenesis, the role of MSCs is invaluable. In addition, without MSCs, hematopoiesis would not be sustained. Therefore, potential toxicity to the MSC system can effect several biological systems simultaneously.

Detection of MSC cytotoxicity is detected using the HemoGenix proprietary MSCGlo-Tox HT Platform. It is specific for MSCs in that the cells are cultured in high-performance MSCGro Medium. Like other HemoGenix ATP bioluminescence assays, MSCGlo-Tox HT is a fully standardized and validated assay platform readout that can be multiplexed with many other assay readouts. This provides a vast amount of data from a single sample.

MSCGlo-Tox HT Multiplexing and Mechanism of Action Studies

• Adipocyte differentiation
• Chondrocyte differentiation
• Osteoblast differentiation
• Flow cytometry to characterize cell populations
• Cell cycle analysis and apoptosis
• Growth factor, cytokine, chemokine production and release
• Apoptosis detecting caspases
• Membrane integrity
• Mitochondrial dysfunction
• Oxidative stress
• Oxidative DNA damage
• Kinase assays
• Protease assays
• Lactate dehydrogenase assays
• Cell-based drug interaction
• Residual toxicity and drug sensitivity change assays

Using ES-derived neural stem and progenitor cells from ArunA Biomedical, HemoGenix has developed NeuroGlo-Tox HT, a 96- or 384-well plate, standardized ATP bioluminescence neurotoxicity screening platform.

ES-Derived Neural Cells for Toxicity Screening

• Human ES (H9, WA09)-derived Neural Progenitor Cells
• Feeder-free
• Serum-free growth
• Stable, diploid karyotype Adherent, proliferating cell line
• Proneural markers: >90% Nestin and Sox 2 positive
• Embryonic marker: <5% Oct-4
• Phenotypic differentiation potential:
• Dopaminergic cells
• Cholinergic cells
• Glutametergic cells
• GABA-ergic cells
• Serotonergic cells
• Astrocytic cells

For more information and a quote, please contact HemoGenix

Drug-induced liver toxicity (DILI) is the most common cause of market drug withdrawals. Hepatotoxicity can result from cellular and mitochondrial damage, oxidative stress and genotoxicity to mention but a few. In addition, drug-drug interactions (DDI) as a result of induction or inhibition of cytochrome P450 enzymes can play a significant role potential toxicity.

HepatoGlo-Tox HT is the proprietary, multiplexing assay platform used by HemoGenix to determine potential cytotoxicity to hepatocytes in vitro. It is specific for fresh cryopreserved, individual or pooled hepatocytes, ES or iPS-derived hepatocytes and hepatocyte cell lines such as Hep G2. It can also be used for 2D- and 3D-hepatocyte models.

HepatoGlo-Tox HT is a fully standardized and validated ATP bioluminescence hepatotoxicity readout that incorporates HepatoGro medium.

Assays that can be Multiplexed with HepatoGlo™-Tox HT

• Cytochrome P450 enzyme assays.
• Cellular drug-drug interaction assays.
• Membrane integrity: LDH or PI dye exclusion.
• Apoptosis: Biochemical caspase detection.
• Mitochondrial dysfunction
• Oxidative stress
• Oxidative DNA damage
• Genotoxicity

In addition to hemotoxicity, immunotoxicity, hepatotoxicity MSC toxicity and neurotoxicity screening and testing, HemoGenix has developed the ComparaTOX Platform that compares and ranks compound toxicity to cells from various organs and tissues. These include cells from the:

• Bladder
• Brain (ES-derived neural stem and progenitor cells for neurotoxicity testing)
• Breast
• Eye
• Heart (e.g. iPS-derived cardiomyocytes)
• Kidney
• Lung
• Liver (primary and iPS-derived hepatocytes for hepatotoxicity testing)
• Ovary
• Prostate
• Skin

The ComparaTOX Platform also includes different transformed cell lines such as the NCI toxicity panel.

For more information, please contact HemoGenix.