Michael Badowski

The potential of cord blood stem cells for use in regenerative medicine

It is estimated that up to 128 million individuals might benefit from regenerative medicine therapy, or almost 1 in 3 individuals in the US. If accurate, the need to relieve suffering and reduce healthcare costs is an enormous motivator to rapidly bring stem cell therapies to the clinic. Unfortunately, embryonic stem (ES) cell therapies are limited at present by ethical and political constraints and, most importantly, by significant biologic hurdles. Thus, for the foreseeable future, the march of regenerative medicine to the clinic will depend on the development of non-ES cell therapies. At present, non-ES cells easily available in large numbers can be found in the bone marrow, adipose tissue and umbilical cord blood (CB). Each of these stem cells is being used to treat a variety of diseases. This review shows that CB contains multiple populations of pluripotent stem cells, and can be considered the best alternative to ES cells. CB stem cells are capable of giving rise to hematopoietic, epithelial, endothelial and neural tissues both in vitro and in vivo. Thus, CB stem cells are amenable to treat a wide variety of diseases including cardiovascular, ophthalmic, orthopedic, neurologic and endocrine diseases.

Comparison of human mesenchymal stem cells derived from adipose and cord tissue.

BACKGROUND AIMS Stem cell therapies can provide an alternative approach for repair and regeneration of tissues and organs. Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies. Although bone marrow-derived MSCs have multi-lineage differentiation potential, bone marrow is not an optimal source because of the isolation process and low yield. The goal of this study was to investigate comparatively for the first time the in vitro regenerative potential of human MSCs from two other sources: umbilical cord tissue and adipose tissue. METHODS Cells from each tissue were isolated with 100% efficiency and characterized by fluorescence activated cell sorting (FACS) analysis for CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105. Growth characteristics were investigated by population doublings, saturation density and plating efficiency. MSCs derived from both types of tissues were assessed for differentiation potential qualitatively and quantitatively. RESULTS FACS analysis showed no differences in expression of CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105 between cord tissue MSCs (CT-MSCs) and adipose tissue MSCs (AT-MSCs). CT-MSCs showed more proliferative potential than AT-MSCs. When cultured in low numbers to determine colony-forming units (CFUs), CT-MSCs showed less CFUs than AT-MSCs. Cells from both sources efficiently differentiated into adipose, bone, cartilage and neuronal structures as determined with histochemistry, immunofluorescence and real-time reverse transcriptase polymerase chain reaction. CONCLUSIONS MSCs can easily be obtained from umbilical cord and adipose tissues, and it appears that both tissues are suitable sources of stem cells for potential use in regenerative medicine.

Cryopreservation of whole adipose tissue for future use in regenerative medicine

BACKGROUND Human adipose tissue (AT) is an ideal stem cell source for autologous cell-based therapies. The preferred setting for tissue engineering and regenerative medicine applications is the availability of clinically acceptable off-the-shelf cells and cell products. As AT is not always available for use, cryopreserved tissue represents an alternative approach. The aim of the present study was to compare the different properties of mesenchymal stem cells (MSCs) isolated from cryopreserved AT. We have measured cell recovery, viability, phenotype, proliferative potential, and differentiation into mesenchymal (adipogenic, osteogenic, chondrogenic) and nonmesenchymal (neuron-like cells) lineages. MATERIALS AND METHODS AT (n = 10) was harvested from donors and either processed fresh or cryopreserved in liquid nitrogen dewars. Both fresh and thawed tissues were enzymatically digested. MSCs were analyzed by fluorescence-activated cell sorting for CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90, and CD105 expression. Growth characteristics of both groups were investigated for population doublings, doubling time, saturation density, and plating efficiency. MSCs derived from fresh and thawed tissues were assessed for differentiation potential both qualitatively and quantitatively. RESULTS Adherent cells from fresh and thawed tissues displayed similar fibroblastic morphology. Cryopreservation did not alter expression of phenotypic markers. Similarly, the proliferative potential of MSCs was not compromised by cryopreservation. Furthermore, cryopreservation did not alter the differentiation capability of MSCs as determined with histochemistry, immunofluorescence, and real time reverse transcriptase-polymerase chain reaction. CONCLUSIONS We conclude that human AT could be successfully cryopreserved for future clinical application and the recovered MSCs were equivalent in functionality to the freshly processed MSCs.

Mixed effects of long-term frozen storage on cord tissue stem cells.

BACKGROUND AIMS Cord tissue (CT) storage is promoted as an opportunity to preserve a source of mesenchymal stromal cells (MSCs) for future use. We analyzed maximal MSC yields from fresh and frozen CT including functional capacity after long-term cryopreservation as a means of assessing potential utility. METHODS CT was evaluated immediately upon harvest or frozen and banked for 5 years before analysis. Upon thawing, cell viability and yield were determined, as were growth characteristics and the ability to differentiate into various tissues. After thawing, enzymatic digestion of CT to release MSCs resulted in poor cell recoveries and few viable cells, requiring explant cultures to recover sufficient cell numbers for analysis. Upon expansion of surviving cells, fluorescence-activated cell sorter analysis showed the cells to be MSCs based on phenotype (CD34-, 45-, 44+, 90+, 105+) and function (ability to form adipocytes and osteoblasts). Frozen CT, however, exhibited decreased plating efficiency, increased doubling times but near equivalent maximum cell expansion, compared with fresh CT. CONCLUSIONS Poor cell yields and recoveries, along with slower growth characteristics, make frozen CT a less-than-optimal choice for MSC banking, despite good functional recovery. In addition, because the amount of fresh CT available at birth is limited and total MSC yields are low, even fresh CT-MSC requires extensive in vitro expansion before clinical use, which limits it application.

Utility of cryopreserved umbilical cord tissue for regenerative medicine.

MSCs can be isolated from adult sources such as bone marrow and adipose tissue. In contrast to these adult tissue sources, harvesting MSCs from cord tissue is a non-invasive procedure and poses no risk to the donor. Stem cell banks offer the opportunity to cryopreserve cord tissue as a source of MSCs for future autologous or allogeneic stem cell based regenerative medicine applications. There is little published data however, characterizing MSCs isolated from cryopreserved cord tissue. The goal of this study was to determine if MSCs isolated from cryopreserved cord tissue are functionally equivalent to MSCs isolated from fresh cord tissue. Umbilical cords were collected from 10 donors. Cords were segmented into 4-6 inch pieces and either cryopreserved or used immediately. Fresh and thawed cord segments were cultured in 7-14 days for outgrowth of MSCs. MSCs were analyzed by FACS for CD45, CD73, CD90 and CD105 expression. FACs analysis confirmed cells isolated from both fresh and frozen tissue expressed MSC markers. Adherent cells were obtained from both fresh and cryopreserved cord tissue segments at a similar plating efficiency. There was no difference in either the number or time of population doublings. MSCs isolated from fresh and frozen tissue were capable of differentiating along adipogenic, chondrogenic, osteogenic and neurogenic pathways, as confirmed by histology and RT-PCR analysis of tissue specific mRNAs. No significant functional differences were observed between MSCs from frozen cord tissue as compared to fresh cord tissue. Cryopreserving cord tissue allows for isolation of MSCs at the point of care when the specific clinical application is known. This may be advantageous as MSC isolation protocols continue to be optimized dependent on intended use.

Collection, Processing, and Banking of Umbilical Cord Blood Stem Cells for Transplantation and Regenerative Medicine

Collection and banking of umbilical cord blood can provide a virtually unlimited source of ethnically diverse stem cell donors. It can be used in place of bone marrow or peripheral blood stem cells for hematologic transplants as well as in a variety of regenerative medicine applications. In this study, we review the latest developments in cord blood banking. We have banked over 300,000 collections at our facility, which were processed by either Ficoll or AXP methodologies. An average 95-99% processing efficiency was obtained. Processed samples can be frozen in either cryovials or bags and banked in the vapor phase of a liquid nitrogen dewar for prolonged periods of time. In conclusion, it is possible to simply and reproducibly harvest, process, and bank cord blood samples using currently available technology.

Subcutaneous Adipose Tissue–Derived Stem Cell Utility Is Independent of Anatomical Harvest Site

Abstract One of the challenges for tissue engineering and regenerative medicine is to obtain suitably large cell numbers for therapy. Mesenchymal stem cells (MSCs) can easily be expanded in vitro to obtain large numbers of cells, but this approach may induce cellular senescence. The characteristics of cells are dependent on variables like age, body mass index (BMI), and disease conditions, however, and in the case of adipose tissue–derived stem cells (ASCs), anatomical harvest site is also an important variable that can affect the regenerative potential of isolated cells. We therefore had kept the parameters (age, BMI, disease conditions) constant in this study to specifically assess influence of anatomical sites of individual donors on utility of ASCs. Adipose tissue was obtained from multiple anatomical sites in individual donors, and viability and nucleated cell yield were determined. MSC frequency was enumerated using colony forming unit assay and cells were characterized by flow cytometry. Growth characteristics were determined by long-term population doubling analysis of each sample. Finally, MSCs were induced to undergo adipogenic, osteogenic, and chondrogenic differentiation. To validate the findings, these results were compared with similar single harvest sites from multiple individual patients. The results of the current study indicated that MSCs obtained from multiple harvest sites in a single donor have similar morphology and phenotype. All adipose depots in a single donor exhibited similar MSC yield, viability, frequency, and growth characteristics. Equivalent differentiation capacity into osteocytes, adipocytes, and chondrocytes was also observed. On the basis of results, we conclude that it is acceptable to combine MSCs obtained from various anatomical locations in a single donor to obtain suitably large cell numbers required for therapy, avoiding in vitro senescence and lengthy and expensive in vitro culturing and expansion steps.

Fusion of HepG2 cells with mesenchymal stem cells increases cancer-associated and malignant properties: An in vivo metastasis model

In the present study, we have tested the hypothesis that fusion between an altered cell and a mesenchymal stem cell produces a hybrid cell with enhanced characteristics associated with metastatic cancer cells, and we have developed a flexible model for investigating the mechanisms of metastasis. Human HepG2 cells with low metastatic potential were induced to fuse with rat bone marrow mesenchymal stem cells, and the progeny were compared with the parental cells for possession of enhanced in vitro and in vivo characteristics of malignant cells. Compared to the parental cells, the fused cells exhibited enhanced expression of E-cadherin, vimentin, Twist, Snail, matrix metalloproteinase 2 and 9 activities, aneuploidy and enhanced in vitro invasion and migration. In an in vivo xenograft assay, the fused cells generated increased numbers of metastatic liver and lung lesions. This model system is a flexible tool for investigation of the mechanisms of stem cell fusion in carcinogenesis and metastasis and for the discovery of new therapeutic targets to inhibit metastasis.

Effect of mild heat stress on the proliferative and differentiative ability of human mesenchymal stromal cells

BACKGROUND AIMS Mesenchymal stromal cells (MSCs) are an attractive candidate for autologous cell therapy, but regenerative potential can be compromised with extensive in vitro cell passaging. Development of viable cell therapies must address the effect of in vitro passaging to maintain overall functionality of expanded MSCs. METHODS We examined the effect of repeated mild heat shock on the proliferation and differentiation capability of human adipose-derived MSCs. Adipose tissue MSCs were characterized by means of fluorescence activated cell sorting analysis for expression of CD3, CD14, CD19, CD34, CD44, CD45, CD73, CD90 and CD105. Similarly, the expression of SIRT-1, p16(INK4a) and p21 was determined by means of polymerase chain reaction. Measurements of population doubling, doubling time and superoxide dismutase activity were also determined. Differentiation of expanded MSCs into bone and adipose were analyzed qualitatively and quantitatively. RESULTS The strategy led to an increase in expression of SIRT-1 concomitant with enhanced viability, proliferation and delayed senescence. The stressed MSCs showed better differentiation into osteoblasts and adipocytes. CONCLUSIONS The results indicate that mild heat shock could be used to maintain MSC proliferative and differentiation potential.

Cell-based therapy for epithelial wounds

BACKGROUND AIMS Bone marrow-derived cells (BMDC) form a significant portion of regenerating epithelial tissue. The purpose of this study was to determine whether exogenous BMDC (containing stroma, stem and progenitor cells), introduced systemically or within the injury site, could enhance the injury repair response. METHODS Excisional wounds (10-mm diameter) were treated by systemic (intravenous; i.v.) and local (subcutaneous; s.c.) administration of BMDC (10-20 × 10(6)/100 μL phosphate-buffered saline). Young and aged BMDC and recipients were studied. RESULTS Young BMDC (2 months old) increased the healing rate compared with older BMDC (1 year old), as measured by the rate of healing and the percentage of healed tissue. Young recipients had statistically better healing efficiency than older recipients. When old BMDC were used, young recipients had a better healing ability than older recipients. In addition, when the size of the healed tissue, the area of repigmentation and hair growth at the injury site were compared, young BMDC and young recipients had superior effects compared with old BMDC and old recipients. CONCLUSIONS These results demonstrate that cellular therapy is important for wound healing in older recipients that do not heal significantly without intervention. BMDC injections result in normal healing, indistinguishable from young recipients. Significantly, a single injection into the wound margin is sufficient to reverse the wounding process and promote normal wound healing. Although younger recipients eventually healed without therapy, BMDC injections accelerated the process, reduced scarring and increased hair regrowth. These findings provide insight into the treatment of non-healing epithelial tissue with BMDC.