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Expansion of Umbilical Cord Blood Stem Cells

A Scarce Resource
Umbilical cord blood (UCB) is a valuable, yet limited resource. On average 50 - 100 ml of blood containing stem cells is obtained from the umbilical cord and the placenta after birth.

When used in treatment the following applies: The greater the number of umbilical stem cells used, the better the prospects for healing. Currently, this is the reason the entire umbilical cord blood specimen is always used in transplantation.

Scientists are conducting research on the expansion of stem cells to increase treatment applications. This means that specimens which today are too small for transplantation could then be used by increasing the number of stem cells.

Worldwide Research
The expansion of umbilical cord blood stem cells at differing stages of maturity has been successfully repeated in research in recent years. Depending on the composition of the experiment, an expansion factor of 10 to more than 1,000 has been achieved. What is most important however, is not the expansion of all (both differentiated and undifferentiated) cells in the cord blood, but most importantly, the expansion of undifferentiated stem cells. After each successful expansion extensive testing, therefore, must be conducted to verify which type of cell actually has expanded and what effect this has had on the cord blood stem cell specimen.

Currently, scientific research is concentrating on umbilical stem cell expansion in three core fields: cellular biology, the medium mix and process technology.

Understanding the Background
What causes stem cells to expand? This is one of the key questions for scientists. The Jülich Research Center, Germany, has, for example, developed a bioreactor , the core of which is formed by small porous spheres made of collagen. Stem cells can settle (Fig. 1) and expand in the hollow spaces of the collagen spheres just as in the niches of bone marrow. The collagen spheres are subsequently dissolved through the addition of an enzyme, allowing the stem cells to be harvested.

Collagen spheres such as those used in the Jülich bioreactors²

Another stem cell expansion study has been conducted by scientists in the U.S. Scientists developed special culture conditions in which a molecule named Delta-1 is critical . This signal molecule formed by the body is involved in the control of cell development. Through treatment with Delta-1 the scientists increased the number of the least mature stem cells one-hundred-fold over conventional processes. Animal experiments have shown the culture cells are more effective than cells that have not been cultivated or were cultivated without Delta-1.

The Right Medium
The expansion of stem cells is undertaken by adding cytokines, so-called growth factors, which cause stem cells to expand or differentiate. In this regard there are two challenges: First, the accurate mix of cytokines must be determined for expansion and second, clinical approval by the FDA must be obtained. Many of the cytokines used successfully in research do not have approval as pharmaceuticals for human use. This is, however, the prerequisite for approval of the entire expansion process.

Closed Processing Systems Preferred
A safe and replicable process is required for routine expansion of stem cells. In the industry, several systems and test conditions are currently competing with one another - In many cases the stem cells are still expanded in culture dishes in an open processing system. These processes are not only extensive; the implementation as a good manufacturing process (GMP), which ensures safety for the patient and required by the FDA, is more difficult.

A closed processing system is, therefore, advantageous in a clinical approval process. Such a system already exists, for example, from the U.S. company, Aastrom Biosciences. In this closed processing system the stem cells are expanded through the addition of a set medium (therapy kit). This expansion system is currently undergoing Phase III clinical trials in the U.S. and is CE certified in Europe. Another alternative is offered by CellGenix, a German company. In this system a special bag is used for stem cell cultures, and the stem cells are subsequently expanded by the addition of a medium.

No False Promises
Umbilical cord blood stem cells, which are used in humans, are considered pharmaceuticals. If expanded cord blood stem cells are to be used in treatment, the expansion process requires approval by the FDA. This, however, will only be granted if clinical studies document that the expanded umbilical cord blood stem cells are just as effective and safe as those that have not been expanded or expanded.

To date, no company in the world has received FDA approval for its expansion system. To date only Aastrom Biosciences has passed a clinical trial with its proprietary system (Table 1).

Table 1: Stem Cell Expansion Companies and Development Status
Provider : Product	Process	Expansion Factor	Market Introduction	Studies	Applications Performed
Aastrom Biosciences (USA): Aastrom-Replicell™	Bioreactor (closed auto-matic system) and therapy kits	No information on CB-I Therapy Kit. CB-II Kit offers 8,3-times more nucleated cells as well as 9.5-times more CD34+ cellsⁱ as compared to CB-I Kit:	Closed system and CB-I Therapy Kit in Phase III Study and CE certified in Europe	Phase I/II Study on expansion of UCB SCⁱⁱ: •Completed for CB I-Kit •Ongoing for CB II-Kit Phase III Study for expanding UCB SC: •Ongoing for CB I-Kit	Children and adults with leukemia and other blood diseases, severe osteopetrosis, lymphomaⁱⁱⁱ
CellGenix (Germany): VueLife® and CellGro®	VueLife® culture bag and CellGro® Medium	No data	VueLife® culture bag is CE-certified. Medium only intended for research	Unknown	Unknown
Cytomatrix (USA): The Cytomatrix™	Cytomatrix™ Bioreactor (Frame for cell growth)	2- 4-fold expansion of UCB SCⁱⁱⁱ	Cytomatrix™ Bioreactor only used clinically but not commer-cially	Expansion of different cell types with the Cytomatrix™ Bioreactor has been described numerous times in scientific literature.	Unknown
Gamida (Israel): StemEx™	Use of low molecular Chelate for copper binding	100-1000-fold expansion of stem and precursor cells within several weeksⁱ	2006ⁱ	Phase I Study for expansion of UCB SC •Study initiated 02/2003	10 persons age 55 with leukemia, non Hodgkin’s lymphoma, Hodgkin’s lymphoma^iv
MainGen (Germany): GMP-Expansion	Culture system	18-fold expansion of stem cells within 7 daysⁱ	Production permit for stem cell expansion received (01/2001)	Pre-Clinical Study on expansion of UCB SC: •Completed Clinical Study on expansion of UCB SC: • Plannedⁱ	Unknown
ViaCell (USA): Selected Amplifica-tion™	Culture medium, removal of differentiated cells	Up to 40-fold expansion of stem cell populationsⁱ	Unknown	Pre-Clinical Study on the expansion of UCB SC: •Completed Clinical Studies on expansion of UCB SC: •Launched end of 2003	None

ⁱManufacturer Information ⁱⁱUmbilical Cord Blood Stem Cells ⁱⁱⁱPreliminary Findings Published ^ivPlanned

Very Promising Results
The successful transplantation of expanded stem cells both from bone marrow as well as from umbilical cord blood has already been documented many times in studies.

For example, U.S. physicians conducted a Phase 1 study on the expansion of umbilical cord stem cells with the AastromReplicell™ system from August 1997 to September 1998. In this study the umbilical stem cell count was increased by a factor of 2.4 and the number of colony forming cells (CFU-GM) was increased 82-fold. Forty-seven (47) months following the transplant the patient’s physician certified that was a permanent engraftment of the stem cells .

Bone marrow stem cells were successfully expanded using the Aas-tromReplicell™ system in 1997 and were then subsequently transplanted. In the Aastrom Bioscience clinical trial the stem cells were cryo-preserved and then thawed and expanded in the bioreactor. In all cases the expanded stem cells regenerated the immune system following chemotherapy. In 1999 in Düsseldorf, Germany, university researchers utilized expanded stem cells in combination with non-expanded stem cells ex vivo (outside the body) .

Based on current knowledge it does not matter whether the stem cells are expanded before or after freezing, which has been confirmed by Italian researchers . This research demonstrated that the freezing process had no negative effects on the expansion capability of the stem cells.

The Future
New information is published on a daily basis promising improved results in the expansion of stem cells. It can be expected that in the coming years additional processes will be developed, each having advantages and disadvantages. These processes will also require approval by regulating bodies; e.g., FDA, before they can be used routinely in treatment. Today, it would be premature to choose which process will be success-ful and secure the required approvals.

For this reason, CorCell will continue its cooperation with companies that have the best suited and currently approved processes.

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Sources:

¹ Research Center Jülich: Blood Stem Cells from the Bioreactor. Press release May 17, 2001.
² Ohishi K et al.: Delta-1 enhances marrow and thymus repopulating ability of human CD34+ CD38–cord blood cells. Clin. Invest. 110:1165-1174 (2002)
³ Jaroscak J et al: Augmentation of um-bilical cord blood transplantation with ex-vivo expanded UCB cells: results of a phase I trial using the Aastrom Replicell system. Blood (2003)
⁴ Bachier CR et al.: Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer. Exp Hematol 27:615-23 (1999)
⁵ Chabannon C et al.: Production of ex vivo expanded hematopoietic cells and progenitors in a closed bioreactor, starting with a small volume marrow collection: A feasibility study in patients with poor-risk breast cancer and receiving high-doses of cyclophosphamide. Int J Oncol 15: 511-518 (1999)
⁶ Kögler, G. et al.: Simultaneous cord blood transplantation of ex vivo expanded together with non-expanded cells for high risk leukemia; Bone Marrow Trans 24: 397-403 (1999)
⁷ Lazzari, L. et al.: Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood; Bone Marrow Trans 28: 693-698 (2001)