Systems and methods for converting adipose-derived mesenchymal stem cells into hematopoietic stem / progenitor cells and differentiating them into blood cells, and applications thereof

Abstract

A method for producing hematopoietic stem / progenitor cells (HS / PCs) and blood cells from adipose-derived mesenchymal stem cells (Ad-MSCs), comprising obtaining the target biological cells, expanding the biological cells, obtaining a pure Ad-MSC cell line from the expanded biological cells, converting the pure Ad-MSC cell line into HS / PCs in vitro, expanding the converted HS / PCs in vitro, and obtaining the expanded HS / PCs.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED PATENT APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 451,269, filed March 10, 2023, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to the field of biotechnology, and more particularly to systems and / or methods for converting adipose-derived mesenchymal stem cells (Ad-MSCs) into hematopoietic stem / progenitor cells (HS / PCs) and differentiating them into blood cells. [Background technology]

[0003] The description of the background art provided herein is intended to generally illustrate the technical background of the present invention. The subject matter discussed in the background art section is not deemed to be prior art solely by virtue of its inclusion in the background art section. Similarly, the problems mentioned in the background art section or problems related to the subject matter of the background of the present invention should not be deemed to have been previously recognized in the prior art. The subject matter in the background art section merely represents different approaches and may be inventions in their own right.

[0004] Recent advances in stem cell technology using human pluripotent stem cells (hPSCs) and multipotent mesenchymal stem cells (MSCs) are opening new doors for patients suffering from previously untreatable diseases and disorders. These cells were first discovered in mouse bone marrow (BM) by Arnold L. Caplan and colleagues. Subsequently, a protocol for directly culturing a subpopulation of stromal cells from bone marrow in vitro was established. Since then, MSCs have been found and derived from various human tissue sources, such as adipose tissue (AT), umbilical cord (UC), umbilical cord blood, placenta, dental pulp, and amniotic fluid. Of these, MSCs derived from adipose tissue, bone marrow, and umbilical cord have been the most studied in human clinical trials. The percentage of MSCs in bone marrow mononuclear cells ranges from 0.001% to 0.01% after gradient centrifugation. The number of MSCs in adipose tissue is at least 500 times greater than in bone marrow, with approximately 5,000 MSCs per gram of adipose tissue. To date, 1,426 registered clinical trials are using MSCs for therapeutic purposes in various trial phases. As supported by a large body of preclinical research and progress in conducting clinical trials, MSCs have proven effective in treating numerous diseases, including nervous system and brain disorders, pulmonary diseases, cardiovascular conditions, wound healing, etc.

[0005] Human adipose tissue was initially considered a passive energy reservoir, but has since been recognized as a major site for sex hormone metabolism, a site of endocrine factor production (e.g., adipsin and leptin), and a source of bioactive peptides known as adipokines. It is now clear that adipose tissue functions as a complex and highly active metabolic and endocrine organ that regulates diverse biological functions. In addition to adipocytes, adipose tissue contains hematopoietic-derived progenitor cells, connective tissue, neural tissue, stromal cells, endothelial cells, MSCs, and pericytes. MSCs are key components of the bone marrow niche, where they interact with hematopoietic stem cell progenitor cells (HSPCs) by providing physical support and secreting soluble factors that control the maintenance and fate of HSPCs. Due to their supportive properties, MSCs have been used to promote hematopoietic stem cell (HSC) engraftment in phase I / II clinical trials of hematopoietic stem cell transplantation (HSCT). Furthermore, MSCs have also been utilized to co-culture and expand HSCs ex vivo prior to medical applications.Recently, several studies have demonstrated that MSCs exhibit broad and potent immunomodulatory capabilities both in vitro and in vivo, which has encouraged their medical applications, particularly in the implementation of hematopoietic stem cell transplantation (HSCT) for the treatment of immune-mediated complications.

[0006] Adipose tissue is composed of lipid-filled mature adipocytes and other non-adipocytes (called stromal vascular fraction (SVF)). SVF is a cell population consisting of various cell types, including stem cells, which are not fully understood. The multipotentiality of stem cells makes these cells (MSCs) promising for tissue engineering and cell therapy. MSCs differentiate relatively easily into chondrocytes and osteocytes under the influence of appropriate growth factors and microenvironmental conditions. However, the differentiation potential of these cells depends on many factors, including the age and sex of the donor and the anatomical location of the fat. AD-MSCs isolated from mice were used as feeders to cultivate mouse-derived Lin-Sca-1 cells. + c-kit +Ad-MSCs were cultured with HSPCs (HSPCs) for 2 and 5 weeks. In vitro studies have shown that Ad-MSCs highly express Jagged-1 and promote LSK cell proliferation, and in vivo studies have shown that Ad-MSCs promote hematopoietic recovery and improved survival in NOD / SCID mice. Previous studies have estimated that the amount of HSPCs in total adipose tissue corresponds to approximately 0.2% of the amount in bone marrow. Can Ad-MSCs actually be used as an alternative source of HSPCs? To date, large-scale conversion of Ad-MSCs into functional HSPCs for basic research and medical applications in vitro has not been successful.

[0007] Human induced pluripotent stem cell (iPSC) technology was pioneered by Shinya Yamanaka and Kazutoshi Takahashi. In 2006, they demonstrated that the introduction of four specific genes encoding transcription factors (Myc, Oct3 / 4, Sox2, and Klf4) could convert somatic cells into pluripotent stem cells. However, reprogramming cells into iPSCs still faces challenges, including low efficiency, genome insertion, and tumorigenicity. Since this breakthrough, iPSC generation and culture systems have significantly improved in terms of transgene introduction (footprint-free), reprogramming conditions (feeder-free, xeno-free), and large-scale production and differentiation of iPSCs, facilitating their advancement in medical applications. iPSC-derived blood products are promising, and several iPSC blood derivatives (e.g., T cells / platelets) have entered clinical trials; however, many challenges remain for many blood lineages. Currently, different protocols and procedures are employed by different laboratories, making comparisons difficult, which in turn limits progress to some extent. Cell and gene therapy has made significant advances during the 21st century. Precision cell therapy based on autologous cells derived from a patient's own body holds great potential for personalized medicine. Indeed, the field is expected to be transformative. However, precision autologous cell therapy still faces significant challenges due to a lack of reliable sources, heterogeneity, and scalability issues. In one aspect, hematopoietic stem cells (HSCs) are extremely difficult to expand in vitro, further complicated by immunogenicity and low transgenic efficiency.

[0008] Currently, the most commonly used hematopoietic stem cell therapy is autologous or allogeneic bone marrow transplantation. However, this treatment is often unsuccessful due to the lack of autologous bone marrow status and / or compatible allogeneic bone marrow sources, as well as side effects associated with allogeneic bone marrow transplantation, such as graft-versus-host disease (GVHD). Considering GVHD, a serious complication that can occur after allogeneic stem cell transplantation, and the high demand for autologous hematopoietic stem cells, finding other stem cell sources would be a real breakthrough and is highly needed in clinical practice.

[0009] Adipose-derived mesenchymal stem cells (Ad-MSCs) share many similarities with mesenchymal stem cells derived from other sources, such as bone marrow, but are harvested from adipose (fat) tissue. Ad-MSCs possess a unique transcriptome and have demonstrated long-term self-renewal, continuous cloning, and multigerm layer differentiation capabilities. Indeed, in recent decades, Ad-MSCs have been actively applied in regenerative medicine in the areas of bone, fat, liver, nerve, and islet cell regeneration and repair. To date, over 131 clinical trials of Ad-MSCs have been approved by the FDA. Ad-MSCs are an abundant source (10%–30% of body weight) and are readily available. For example, isolation of Ad-MSCs typically involves harvesting adipose tissue via liposuction, which is less invasive and often associated with fewer complications than bone marrow harvesting. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, overcoming the aforementioned deficiencies and inadequacies, particularly the development of AdMSCs, which can be directly extracted from mature adipose tissue, the body's greatest resource, as a new source for inducing hematopoietic stem / progenitor cells (HS / PCs), remains unachieved. Mature adipose tissue not only does not require embryos but can also be produced in a patient-specific manner, meaning that each patient may have their own pluripotent stem cell line. An unlimited supply of these autologous cells can be used to generate grafts without the risk of immune rejection. This could provide a chance of survival for patients with previously untreatable diseases.

[0011] In light of the above, the present invention discloses a method for producing HS / PCs and differentiating adipose-derived mesenchymal stem cells (Ad-MSCs) into blood cells, which includes obtaining target living cells, expanding the living cells, obtaining a pure Ad-MSC cell line from the expanded living cells, converting the pure Ad-MSC cell line into hematopoietic stem / progenitor cells (HS / PCs) in vitro, expanding the converted HS / PCs in vitro, and obtaining the expanded HS / PCs.

[0012] In one embodiment, the method further comprises differentiating the expanded HS / PCs into a blood cell lineage without co-culturing with bone marrow-derived HSCs, the blood cell lineage comprising at least one of white blood cells (WBCs), red blood cells (RBCs), and platelets.

[0013] In one embodiment, the process of converting a pure Ad MSC cell line into HS / PC does not use any exogenous genes.

[0014] In one embodiment, the process of converting a pure Ad MSC cell line into HS / PC does not involve any co-culture with any other cells.

[0015] In one embodiment, obtaining a pure Ad MSC cell line comprises isolating an Ad MSC single cell line from a biological sample and culturing the Ad MSC single cell line.

[0016] In one embodiment, the step of converting a pure Ad MSC cell line into HS / PC comprises using an HS / PC conversion kit.

[0017] In one embodiment, the HS / PC conversion kit comprises Ad MSC basal medium, basal nutritional supplements and a basal conversion supplement mixture.

[0018] In one embodiment, the process of converting a pure Ad MSC cell line into HS / PC includes mixing a mixture of basal nutritional supplements and basal conversion supplements into an Ad MSC basal medium to prepare a HS / PC-C complete medium, adding the HS / PC-C complete medium to the resulting Ad MSC single cell line, and incubating the pure Ad MSC cell line in the HS / PC-C complete medium for a certain period of time.

[0019] In one embodiment, the process of converting a pure Ad MSC single cell line into HS / PC has a conversion efficiency of greater than 10%.

[0020] In one embodiment, the combination of the converted HS / PCs and the enhanced system has a higher gene transfection efficiency (about 25-fold higher than that of the converted HS / PCs or normal HS / PCs derived from bone marrow).

[0021] In one embodiment, the converted HS / PC comprises a gene carry-on cell or gene transfer system.

[0022] In another aspect of the present invention, a HS / PC conversion kit for converting adipose-derived mesenchymal stem cells (Ad-MSCs) into hematopoietic stem / progenitor cells (HS / PCs) comprises an Ad-MSC basal medium, a basal nutritional supplement, and a basal conversion supplement mixture, and the kit converts Ad-MSCs into HS / PCs.

[0023] In one embodiment, the basal nutritional supplement comprises fetal bovine serum and horse serum.

[0024] In one embodiment, the basal conversion supplement mixture includes at least one of insulin, holotransferrin, sodium selenite (ITS solution), L-ascorbic acid, GM-CSF, SCF, VEGF, IGF-I, IGF-II, IL-3, Flt3-L, thrombopoietin (TPO), dexamethasone, fatty acid-free BSA, 1-thioglycerol, SB431542, CHIR99021, and Y-27632.

[0025] In one embodiment, the basal conversion supplement mixture includes insulin, holotransferrin, sodium selenite (ITS solution), L-ascorbic acid, GM-CSF, SCF, VEGF, IGF-I, IGF-II, IL-3, Flt3-L, thrombopoietin (TPO), dexamethasone, fatty acid-free BSA, 1-thioglycerol, SB431542, CHIR99021, and Y-27632.

[0026] In yet another aspect of the present invention, an RBC and platelet differentiation kit for differentiating hematopoietic stem / progenitor cells (HS / PCs) into red blood cells (RBCs) and platelets includes a basal cell culture medium, a basal nutritional supplement, and an RBC supplement mixture, wherein the kit differentiates the HS / PCs into at least one of RBCs and platelets.

[0027] In one embodiment, the basal nutritional supplement comprises fetal bovine serum and horse serum.

[0028] In one embodiment, the RBC supplement mixture includes at least one of insulin, holotransferrin, sodium selenite (ITS solution), L-ascorbic acid, SCF, VEGF, IGF-I, IGF-II, IL-3, Flt3-L, thrombopoietin (TPO), dexamethasone, fatty acid-free BSA, 1-thioglycerol, EPO, ferrous sulfate, PDGF BB, and activin A.

[0029] In yet another embodiment of the present invention, a WBC differentiation kit for differentiating hematopoietic stem / progenitor cells (HS / PCs) into white blood cells (WBCs) comprises a basal cell culture medium, a basal nutritional supplement, and a WBC supplement mixture, wherein the kit differentiates HS / PCs into WBCs.

[0030] In one embodiment, the basal nutritional supplement comprises fetal bovine serum and horse serum.

[0031] In one embodiment, the basal conversion supplement mixture includes at least one of insulin, holotransferrin, sodium selenite (ITS solution), L-ascorbic acid, SCF, VEGF, IGF-I, IGF-II, IL-3, Flt3-L, thrombopoietin (TPO), dexamethasone, fatty acid-free BSA, 1-thioglycerol, BMP4, IL-7, IL-12, IL-11, IL-6, IL-2, FGF-b, TGF-a, TGF-b1, TGF-b2, TGF-b3, IL-1a, IL-1b, IL-4, IL-5, IL-8, IL-10, IL-12, IL-32a, mIL-36R2, and GM-CSF.

[0032] In yet another aspect of the present invention, an automated multi-function system for processing and culturing single cells comprises a housing, at least one small fluid volume dispenser system disposed within the housing, and a tissue disruption system disposed below the small fluid volume dispenser system and within the housing, wherein the small fluid volume dispenser system is configured to dispense at least one fluid and the tissue disruption system receives the at least one fluid.

[0033] In one embodiment, the small fluid volume dispenser system is configured to dispense at least one fluid in a volume of between about 5 mL and 10 mL.

[0034] In one embodiment, the small fluid volume dispenser system includes a cylinder that houses one or more pre-filled cartridges.

[0035] In one embodiment, the small fluid volume dispenser system includes a top needle positioned above one or more pre-filled cartridges and a bottom needle positioned below one or more pre-filled cartridges.

[0036] In one embodiment, the top and bottom needles are configured to move vertically to pierce the filled cartridge.

[0037] In one embodiment, the tissue disruption system includes a vial mechanically connected to a motor.

[0038] In one embodiment, the vial contains a top grinder plate, a bottom grinder plate, and a filter positioned below the top and bottom grinder plates.

[0039] In one embodiment, the top grinder plate and the bottom grinder plate are arranged to form a sample placement site for receiving a biological sample.

[0040] In one embodiment, the tissue disruption system further comprises a tube fluidly connected at a first end to the bottom of the vial.

[0041] In one embodiment, the tube is fluidly connected at the second end to a dispenser funnel.

[0042] In one embodiment, the device comprises a cell flask rotor, the cell flask rotor comprising a plurality of magnetic nanoparticles. [Brief explanation of the drawings]

[0043] The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used in the drawings to refer to the same or like elements in relation to the embodiments. [Figure 1] FIG. 1 illustrates photomicrographs showing Oil Red O staining of human and mouse Ad MSCs. [Figure 2] Figure 2 shows the Ad-MSC culture growth curve. Ad-MSC proliferation increased linearly after 7 days of culture. [Figure 3] FIG. 3 shows photomicrographs demonstrating that proliferation of Ad MSCs increased linearly after 7 days of culture. [Figure 4] FIG. 4 shows markers that were positively expressed in Ad MSCs derived from human breast adipose tissue. [Figure 5]FIG. 5 shows markers that were positively expressed in Ad MSCs derived from abdominal adipose tissue of SS mice. [Figure 6] FIG. 6 shows markers positively expressed in Ad MSCs derived from CD45.1 mice. [Figure 7] FIG. 7 shows the converted HS / PC using UL™ h or m HS / PC-C complete medium for human culture. [Figure 8] FIG. 8 shows the converted HS / PC using UL™ h or m HS / PC-C complete medium for culture on mice. [Figure 9] FIG. 9 shows the results of flow cytometry for sorting Sca-1 positive cells from mouse HS / PCs after conversion. [Figure 10] Figure 10 shows that the markers CD90.2 (blue), cKit (CD117) (purple), CD48 (green), CD105 (red), and CD150 (pink) are positively expressed in converted Sca-1-positive mouse HS / PCs (D). [Figure 11] FIG. 11 shows that the FITC-Sca-1 marker (green) is positively expressed in transformed mouse HS / PC(E) cultured as live cells. [Figure 12] Figure 12 shows the flow cytometry results showing that the markers CD34 (green, red), CD90 (yellow, red), and CD49f (pink) are positively expressed in post-conversion HS / PCs derived from human cells. [Figure 13] FIG. 13 shows that after 18-23 days of culture in UL™ hRBC medium, supernatant cells derived from human converted HS / PCs positively expressed the glycophorin A (CD235a) (green) marker. [Figure 14] FIG. 14 shows flow cytometry results showing 18-23 days of post-conversion human HS / PC cultured in UL™ hRBC complete medium. [Figure 15]FIG. 15 shows that supernatant cells from mouse post-conversion HS / PCs positively expressed the glycophorin A (TER 119) (green) marker after 18-23 days of culture in UL™ mRBC medium. [Figure 16] Figure 16 shows flow cytometry results showing converted mouse HS / PCs cultured in UL™ mRBC complete medium after 18-23 days. [Figure 17] FIG. 17 shows the results of flow cytometry demonstrating positive expression of CD45.1 (red) and TER 119 (green) in blood cells of CD45.2 mice transplanted with HS / PCs after conversion of CD45.1 mice on day 23. [Figure 18] FIG. 18 shows the results of flow cytometry demonstrating positive expression of CD45.1 (red) and TER 119 (green) in blood cells of CD45.2 mice transplanted with HS / PCs after conversion of CD45.1 mice on day 23. [Figure 19] FIG. 19 shows the transplantation of post-conversion HS / PCs from CD45.1 mice into six CD45.2 mice. [Figure 20] FIG. 20 shows CBC results showing cells in the supernatant from a 25-day culture of murine post-conversion HS / PCs in UL™ mWBC medium. [Figure 21] FIG. 21 shows the results of CBC showing cells in the supernatant from a 25-day culture of mouse post-conversion HS / PCs in normal medium. [Figure 22] FIG. 22 shows flow cytometry analysis demonstrating positive expression of leukocyte markers in the peripheral blood of CD45.2 mice compared to CD45.1 mice. [Figure 23] FIG. 23 shows that in transformed HS / PCs transfected with pLenti-GFP, there are at least 25 GFP-positive cells (green) after 3 days of culture in UL™ TFK medium. [Figure 24] FIG. 24 shows that in transformed HS / PCs transfected with pLenti-GFP medium, only one GFP-positive cell was present on day 3 of culture under normal medium conditions. [Figure 25] Figure 25 shows a schematic diagram of an automated multi-function system for processing and culturing single cells, with the door closed in the top image and open in the bottom image. [Figure 26] FIG. 26 shows front and side views (top) and a schematic diagram (bottom) of a fluid volume dispenser system for a cartridge-based dispensing system of an automated multi-function system for processing and culturing single cells. [Figure 27] FIG. 27 shows a side view and schematic diagram of the tissue grinder system of an automated multi-function system for processing and culturing single cells. [Figure 28] FIG. 28 shows a schematic, side cross-sectional and exploded view of a vial with a filter screen and grinder plate. [Figure 29] FIG. 29 shows a cross-sectional view of the tissue grinder system of an automated multi-function system for processing and culturing single cells. [Figure 30] FIG. 30 shows a cross-sectional view of the tissue grinder system in the lowered position (left) and with the panel raised (right). [Figure 31] FIG. 31 shows a front view of the cartridge-based dispensing system configuration for an automated multi-function system for processing and culturing single cells. [Figure 32] Figure 32 shows a schematic diagram of the cell flask rotor. Within the cell flask rotor are an array of neodymium magnets that activate magnetic nanoparticles as fluid moves through the cell flask. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention will now be described in more detail with reference to the accompanying drawings, which illustrate exemplary embodiments of the present invention. However, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. Like reference numerals refer to like elements throughout.

[0045] Terms used herein have their ordinary meanings as understood by those skilled in the art within the context of the present invention and are interpreted based on the specific context in which each term is used. Certain terms used to describe the present invention are explained below or elsewhere in the specification to aid those skilled in the art in understanding the description of the present invention. For convenience, certain terms may be highlighted in italics and / or quotation marks. The use of highlighting does not affect the scope and meaning of a term; i.e., the scope and meaning of the same term is independent of the presence or absence of highlighting. It should be understood that the scope and meaning of the same term may be described in multiple ways. Therefore, for any term used herein, alternative expressions or synonyms may be used, and no special significance should be attached to whether or not the term is explained or described in detail. For some terms, synonyms are provided. The use of one or more synonyms does not exclude the use of other synonyms. Examples herein, including the example terms described herein, are for illustrative purposes only and do not limit the scope or meaning of the invention or the exemplified terms. Similarly, the invention is not limited to the various embodiments presented herein.

[0046] Those skilled in the art will understand that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those described can be used in the practice of the present invention without undue experimentation. All art-known functional equivalents of these materials and methods are intended to be within the scope of the present invention. The terms and expressions used herein are for purposes of description and not limitation. Therefore, the use of such terms and expressions is not intended to exclude equivalents of the described features or portions thereof, and it should be recognized that various modifications are possible within the scope of the claims. Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art will be able to modify and change the concepts disclosed herein, and these modifications and changes are intended to be within the scope of the present invention as defined by the appended claims.

[0047] When a range is given herein, such as a temperature range, a time range, or a composition or concentration range, it is intended that all intermediate ranges, subranges, and individual values ​​contained within that range are encompassed within the present invention. Any subrange or individual value within a range or subrange described herein may be excluded from the claims.

[0048] Furthermore, as used herein and in the claims, the terms "a," "an," and "the" are understood to include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a plurality of cells and equivalents thereof known to those of skill in the art. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0049] When an element is referred to as being "on," "attached to," "connected to," "coupled to," "in contact," etc., with another element, it is understood that the element may be directly on, attached to, connected to, coupled to, or in direct contact with the other element, or that intervening elements may be present. In contrast, when an element is referred to, for example, as being "directly on," "directly attached to," "directly connected to," "directly coupled to," or "in direct contact with," there are no intervening elements present. Also, it will be understood by those skilled in the art that references to structures or features that are positioned "adjacent" to another feature may have portions that overlap or are positioned below the adjacent feature.

[0050] Terms such as first, second, and third may be used herein to describe various elements, components, regions, layers, and / or sections, but it is understood that these elements, components, regions, layers, and / or sections are not limited by these terms. These terms are used only to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a first element, component, region, layer, or section discussed below could be referred to as a second element, component, region, layer, or section without departing from the teachings of the present invention.

[0051] Additionally, relative terms such as "lower" or "bottom" and "upper" or "top" may be used to describe a relationship to other elements depicted in the figures. It should be understood that the relative terms are intended to encompass not only the illustrated orientation, but also other orientations of the device. For example, if the device depicted in the figures were inverted, an element described as being "lower" than other elements would then be located "above" such other elements. Thus, the exemplary term "lower" can encompass both an orientation of "lower" and "upper," depending on the particular orientation of the figure. Similarly, if the device in the figures were inverted, an element described as being "below" or "beneath" other elements would now be oriented "above" the other elements. Thus, the exemplary terms "lower" or "beneath" can encompass both an orientation of above and below.

[0052] It is further understood that the terms "comprises" and / or "comprising," or "includes" and / or "including," or "has" and / or "having," or "carry" and / or "carrying," or "contain" and / or "containing," or "involve" and / or "involving," "characterized by," and the like are meant to be open-ended, i.e., inclusive, and not limiting. When used in this disclosure, these terms specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0053] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Terms listed in general dictionaries should be interpreted in light of the relevant technical field and the context of this invention, and should not be interpreted in an idealized or overly formal sense unless specifically defined herein.

[0054] As used in this disclosure, "nearly," "about," "approximately," or "substantially" generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical values ​​presented herein are approximate, meaning that the terms "nearly," "about," "approximately," or "substantially" can be inferred even when not explicitly stated.

[0055] As used in this disclosure, the phrase "at least one of A, B, and C" should be interpreted to mean (A or B or C) using a non-exclusive logical OR. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0056] As used in this disclosure, the term "treatment" refers to any protocol, method, and / or agent that may be used in the management, treatment, and / or amelioration of a given disease or its associated symptoms. In certain embodiments, the terms "therapies" and "therapy" refer to biological therapy, supportive therapy, and / or other therapies known to those of skill in the art, such as medical professionals, that are useful in the management or treatment of a particular disease or its associated symptoms.

[0057] As used in this disclosure, "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a given disease resulting from the administration of one or more therapies, including, but not limited to, the administration of the microspheres disclosed herein.

[0058] The inventive system described herein features innovative strategies and sources for inducing HS / PCs, in particular innovative systems and / or methods for converting Ad MSCs into HS / PCs and differentiating them into blood cells.

[0059] This innovative system demonstrates that mature adipocyte-derived MSCs can be converted into HS / PCs, which can then be differentiated into multiple peripheral blood cell types in vitro and in vivo. In one embodiment, the converted hematopoietic stem cells (HSCs) demonstrate high gene transfection efficiency. This system establishes a new source for autologous cell and gene therapy with fewer side effects than existing technologies.

[0060] The present invention discloses a system with four major breakthroughs.

[0061] In one embodiment, the present invention discloses a unique Ad MSC cell culture protocol / reagent for converting Ad MSCs into HS / PCs in vitro. In one embodiment, the present invention creates a conversion and expansion system for converting Ad MSCs into HS / PCs in vitro without introducing any exogenous genes. In one embodiment, the conversion efficiency to HS / PCs is about 11.7%. In one embodiment, the present invention expands the converted HS / PCs in vitro to large scale.

[0062] In one embodiment, the present invention discloses unique protocols / reagents for the differentiation of converted HS / PCs into various peripheral blood cell lineages both in vitro and in vivo. The present invention creates a system for the in vitro and in vivo differentiation of converted HS / PCs into various peripheral blood cells, such as RBCs, platelets, and WBCs.

[0063] In one embodiment, the present invention discloses a unique protocol / reagent for improving the gene transfection system of in vitro converted HS / PCs. The present invention creates a system that improves gene delivery, thereby improving the gene transfection efficiency of converted HS / PCs. In one embodiment, the gene transfection efficiency of the present invention is 25 times higher than that of conventional reagents in existing technologies. In one embodiment, converted HS / PCs are gene carrier cells that provide a guarantee for the success of gene therapy.

[0064] In one embodiment, the present invention discloses an automated bedside single cell isolation system having one or more of the following features: (1) an all-in-one integrated device of centrifugation-free, contamination-free tissue disruption, enzymatic digestion, and nanopurification with one scan-recording system for rapid tissue single cell and specific blood cell isolation; (2) a standardized protocol to ensure high quality of isolated cells; and (3) bedside operation to minimize environmental exposure time and maximize cell culture viability.

[0065] In one embodiment, the present invention of converting Ad MSCs as a new process for inducing HS / PCs in precision cell and gene therapy has one or more of the following advantages and implications:

[0066] In one embodiment, the present invention ensures the safety of the induced HS / PCs and the blood cells produced therefrom. In particular, this innovative system does not require the introduction of any exogenous genes, thus eliminating the concern about contamination with co-emergent tumorigenic stem cells, as seen with other induced or embryonic cells. Furthermore, Ad-MSC-converted HS / PCs are derived from autologous adipose tissue, eliminating the risk of GVHD side effects associated with autologous cell transplantation.

[0067] In one embodiment, the present invention offers resource advantages. Specifically, Ad-MSCs are derived from adipose tissue (Ad), which is widely distributed and widely available compared to the limited resource of MB-MS: M in vivo: 10%-20%, F: 20%-30%. Thus, the conversion rate for Ad-MSCs is approximately 11.7% compared to 1% for BM.

[0068] In one embodiment, the present invention provides a high processing efficiency. In particular, harvesting adipose tissue is convenient and rapid and can typically be obtained by liposuction, minimally invasive surgery, or other surgical procedures, all of which are less invasive and often result in fewer complications than harvesting bone marrow.

[0069] In one embodiment, the present invention is advantageous in terms of culture. In particular, in vitro culture of Ad-MSCs and converted HS / PCs can be efficiently achieved on a large scale for potential clinical needs. Ad-MSCs have a long shelf life compared to other types of stem cells, such as induced pluripotent or embryonic SCs.

[0070] One important advantage of the present invention is that the conversion of Ad MSCs to HS / PCs and the differentiation of HS / PCs into various blood cells are achieved without co-culture with bone marrow-derived HSCs and / or without the insertion of exogenous genes. That is, HS / PCs are produced using a pure Ad MSC cell line, and various blood cells are differentiated from the HS / PCs produced from the pure Ad MSC cell line. Other cells, such as SVF or bone marrow-derived HSCs, have not previously been used in co-culture with Ad MSCs and / or the HS / PCs produced therefrom. A pure Ad MSC cell line is defined as a cell line consisting essentially of or consisting only of Ad MSCs. In practice, as shown in Figure 1, a pure Ad MSC cell line (Oil Red O staining is a special stain for adipose-derived mesenchymal stem cells (Ad MSCs)) is obtained by culturing living cells obtained from a subject.

[0071] In one embodiment, the biological cells are obtained at the bedside, for example, next to the subject from whom the sample is obtained.

[0072] In one embodiment, the present invention is in ethics compliance, in particular, the present invention does not carry the baggage of ethical controversy that embryonic stem cells may carry in some contexts.

[0073] In one embodiment, the present invention is function-based. In particular, the present invention has powerful value-added capabilities, being able to secrete approximately 247 beneficial cell trophic and stimulatory factors, including anti-inflammatory, immunomodulatory, angiogenic, and trophic agents, all of which are beneficial in the context of intensive treatment and regeneration.

[0074] In one embodiment, the present invention is application-based. In particular, the present invention has a wide range of differentiation potential, including gene carrier cells, bone cells, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic cells, hepatocytes, HSCs, progenitor cells, and blood cells. Therefore, the present invention can be used for the regeneration of various organs and tissues.

[0075] Example 1 - Automated multi-function system for processing and culturing single cells In one embodiment, the present invention discloses an all-in-one multifunctional system for processing and culturing single cells, which ensures highly efficient cell isolation and high cell viability, while reducing the cost of cell therapy for hospitals and patients.

[0076] In one embodiment, the system provides one-step isolation of Ad MSCs or tissue and immune cells in approximately 60 minutes without centrifugation, multi-steps and contamination risks.

[0077] In one embodiment, the system provides a well-sealed, sterile, constant temperature system that ensures freedom from contamination and high cell viability.

[0078] In one embodiment, the system provides standardized operation and one scan data record, which ensures confidentiality and long-term tracking of data.

[0079] In one embodiment, the system is portable and supports bedside use, which ensures widespread application.

[0080] In one embodiment, this system overcomes current challenges in cell processing by maximizing cell viability, minimizing cell contamination, and reducing costs. This system offers rapid, portable, and widespread application to increase the effectiveness and availability of cell-based treatments.

[0081] In one embodiment, an apparatus for isolating viable cells from tissue or blood samples includes one or more of the following components: an intake designed to receive tissue samples or surgically removed tissue directly from a syringe to ensure minimal handling and reduce contamination risk; a tissue grinder tube and / or robotic rotary shaker (Part A) to facilitate efficient isolation of viable cells from tissue; a cell culture flask containing approximately 5 mL to 10 mL of nanoparticle-specific antibody solution (Part Ab); a robotic pipette arm with multiple nozzles to dispense cell suspensions and other solutions to transfer and wash cells (Part B); a shakeable magnetic platform to facilitate adhesion of cells to the bottom of the cell culture flask or conjugation with specific nanoantibodies (Part C); a robotic seal for sealing the flask to facilitate sterile transport of the cell culture flask. a cottered capper arm (Part D); an automatic or semi-automatic lever-type connector mechanism (Part E) that connects all post-sterilization solution cartridges in the sealed container to maintain sterility and prevent direct human handling; an insulated carrier for sealed cell culture flasks (Part F) with temperature control at 37°C and CO2 supplementation to increase the number of viable cells in transport from the patient's bedside operation to the laboratory for further cell processing (the device should remain at a constant 37°C when powered on); multiple germicidal UV+ozone sources (Part G) located throughout the system to ensure sterility within the system; and a barcode scanner (Part H) that registers sample tissues with the device before processing and associates them with tagged cell culture flasks for traceability and compliance with medical documentation practices.

[0082] In one embodiment, the operator fills a grinder vial with a tissue sample and then places the vial onto the bottom adapter of the grinder subassembly.

[0083] In one embodiment, the operator then operates the over-center mechanism to raise the grinder vial towards the grinder gearbox.

[0084] In one embodiment, the bottom and top seals of the grinder are pierced to couple the vial to the machine.

[0085] In one embodiment, 5 mL of buffer is dispensed into the grinder to facilitate separation of cells from the tissue.

[0086] In one embodiment, after a set time has elapsed, an additional 5 mL of buffer is dispensed into the grinder and negative pressure is applied via a peristaltic pump, the outlet of which is connected to the cell flask.

[0087] In one embodiment, the isolated cells are washed with saline in a cell flask and then magnetic nanoparticles are added.

[0088] In one embodiment, after several washes, buffer is applied to the cell flask and then the flask is sealed in preparation for shipping.

[0089] In one embodiment, the present invention discloses a small fluid volume dispenser system. The purpose of this system is to aseptically dispense small volumes of fluid, specifically 5 mL to 10 mL. Such dispensing is important for tasks requiring precision and cleanliness, such as those in cell biology or medical research.

[0090] In one embodiment, the present invention discloses a cartridge-based dispensing process. Instead of using pumps, tubes, or reservoirs that can compromise sterility, the system uses pre-filled cartridges. Each cartridge contains a precise amount of fluid. A set of needles punctures both the top and bottom of the cartridge to facilitate fluid flow. The top needle is actuated to create a vent and to push the cartridge toward the bottom needle for ejection.

[0091] In one embodiment, the present invention discloses a hydrophobic funnel. Specifically, when a fluid is dispensed, it passes through a hydrophobic funnel, which repels water, ensuring that the fluid flows smoothly into the desired container without contamination or loss of product.

[0092] Advantages of this system include eliminating the need for measurement sensors and positive displacement pumps, simplifying the system, and reducing cell buffer loss, which are important for minimizing waste and cleaning.

[0093] Figure 25 shows a schematic diagram of an automated multi-function system for processing and culturing single cells. The top schematic shows the door closed, and the bottom schematic shows the door open.

[0094] FIG. 26 shows front and side views (top) and a schematic diagram (bottom) of a fluid volume dispenser system for a cartridge-based dispensing system of an automated multi-function system for processing and culturing single cells.

[0095] In particular, one or more cartridges 1 are disposed inside a cylinder 4. A spring may be disposed below the cartridge to push the cartridge up against a cylinder cap 11 disposed on top of the cylinder. The cartridge 1 may be connected at its bottom to a bottom discharge needle 10. A cylinder rod 7 passes through the center of the cylinder 1. A motor 2 is supported on a bracket 5 and disposed above the cylinder 4. The motor 2 is connected to and drives the cylinder rod 7. Above the cartridge 1, a linear actuator 3 is connected to a needle extension that receives an upper vent needle 9. In one embodiment, there is a needle holder 12 that is in direct contact with the vent needle 9.

[0096] FIG. 27 shows a side view and schematic diagram of the tissue grinder system of an automated multi-function system for processing and culturing single cells.

[0097] In particular, in one embodiment, the tissue grinder system includes a 30 mL vial with a 70 nm filter screen and grinder plates. During operation, a tissue sample is placed inside the vial between a pair of grinder plates. The system compresses the tissue between the two grinder plates and seals with a screw cap.

[0098] For fluid dispensing, 5 mL of buffer is dispensed into the vial to aid in disruption. This dispensing is performed inside the cell isolation machine to ensure sterility.

[0099] In one embodiment, the vial is placed on the adapter and raised to engage the gears that drive the fracturing action. A needle funnel that receives the dispensed fluid also pierces the seal for sterility.

[0100] In one embodiment, in a post-disruption process, an additional 5 mL of fluid is added to rinse the sample after disruption.

[0101] In one embodiment, a peristaltic pump applies negative pressure to collect the suspension cells, which are then transferred to a cell flask for further processing, maintaining a sterile environment throughout the entire process.

[0102] The system aims to process biological samples in a sterile and efficient manner, minimizing the risk of contamination and ensuring the integrity of the sample for research or medical purposes.

[0103] FIG. 28 shows a schematic, side cross-sectional and exploded view of a vial with a filter screen and grinder plate.

[0104] In one embodiment, the vial includes a screw cap 15 that receives a plug 16. A top induction seal 17 is received in the plug 16. A top grinder plate 18 and a bottom grinder plate 19 are mated to each other. A filter screen 20 in a filter holder 21 is positioned below the top grinder plate 18 and the bottom grinder plate 19. A vial 25 mates with the screw cap 15 to form a housing that contains the aforementioned parts. A bottom induction seal 22 is attached to the bottom of the vial 25.

[0105] FIG. 29 shows a cross-sectional view of the tissue grinder system of an automated multi-function system for processing and culturing single cells.

[0106] In one embodiment, the tissue grinder system includes a needle funnel 12 with a bottom tube that passes through a through-hole large gear 13 located inside a gearbox. The bottom end of the needle funnel 12 is received inside the vial described above. An induction seal needle drain 23 is fluidly connected to the bottom of the vial 25 and transports fluid through silicone tubing. Silicon tubing 26 is received in an over-center mechanism 28 supported by a bottom adapter 27. Silicon tubing 26 then passes through a peristaltic pump 29 and is fluidly connected to a dispensing funnel 30.

[0107] In one embodiment, the fluid volume dispenser system is located above the tissue grinder system in the device, so that a solution, such as a buffer, is dispensed by the fluid volume dispenser system and passes through the top of the vial screw cap 15 and immediately through the coupling that transfers power to the top and bottom grinder plates 18 and 19, where it is dispensed into the vial 25.

[0108] FIG. 30 shows a cross-sectional view of the tissue grinder system in the lowered position (left) and in the raised panel (right).

[0109] 31 shows a front view of the cartridge-based dispensing system configuration of an automated multi-function system for processing and culturing single cells. In particular, aside from the fluid volume dispenser system positioned above the tissue disruption system, in one embodiment, the system also includes one or more of a UV sterilizing light 31, a capper 32, and a cell flask rotator 33 configured to receive a cell flask 34 and fluidly connected to a drain 35.

[0110] Figure 32 shows a schematic diagram of the cell flask rotor. Within the cell flask rotor are an array of neodymium magnets that activate magnetic nanoparticles as fluid moves through the cell flask.

[0111] Example 2 - Protocol for tissue single cell isolation and Ad MSC culture Protocol for preparing tissue In one embodiment, the present invention discloses a protocol for preparing tissue, which should be appropriately modified based on the weight and / or volume of the tissue to be prepared without departing from the essence of the present invention. 1. To prepare 500 mL of wash buffer pre-warmed to 37° C., add 5.5 mL of 100× antibiotic-antimycotic solution (AA) to 500 mL of wash buffer. 2. To prepare complete mesenchymal stem cell expansion medium, add serum-supplemented B to a 500 mL bottle of Mesenchymal Stem Cell Expansion Medium A (UL™-MSC-E) and 5 mL of 100X antibiotic-antimycotic solution (AA). 3. Place an absorbent pad inside the hood. 4. Add approximately 10 mL of the pre-warmed wash buffer prepared in step 1 to a sterile 100 mm culture dish. 5. Obtain the tissue and place the container containing the tissue in a laminar flow hood. 6. Remove the lid from the 100 mm culture dish and transfer the tissue with sterile forceps into the dish containing the pre-warmed wash buffer. 7. Wash the tissue two or three times in the medium contained in the 100 mm culture dish by stirring with forceps.

[0112] Protocol for the primary isolation of mature adipose or other tissues In one embodiment, the present invention discloses a protocol for the primary isolation of mature adipose or other tissues. 1. Surgically removed tissue is placed directly into Part A of the system of Example 1. Part A automatically disrupts the tissue into single cells using a robotic rotary vibrator. After tissue dispersion, the grinder tube automatically rotates to a vertical position. The cell suspension flows by gravity to the bottom of the grinder tube. The grinder is then washed with 10 mL of UL™ TMSC-IS. 2. Incubate the collected cell suspension with shaking at 37°C for 20-30 minutes. Transfer the entire cell suspension to a new cell culture flask. 3. Place a new cell culture flask flat on Part C and incubate the flask at 37°C for 10 minutes. 4. Aspirate the supernatant according to Part B of the system, then add 10 mL of wash buffer to the flask to wash the cells twice. Add 10 mL to 20 mL (depending on the size of the flask) of UL™ TMSC-ISMSC-E to the flask, and seal the culture flask according to Part D. 5. Transfer the culture flask to an incubator at 37°C and 5% CO2 and incubate for 5 to 7 days.

[0113] Protocol for isolating and culturing Ad-MSCs In one embodiment, the present invention discloses a protocol for isolating and culturing Ad MSCs. 1. Using sterile forceps, place the tissue on the grinder and spread it flat. 2. Transfer the grinder to the cell processing device. 3. Start Program 1 for fat or other tissues. 4. Remove the flask from the processing device and transfer the flask to an incubator and culture at 37°C and 5% CO2 with appropriate humidity.

[0114] Results: 72 hours after seeding the primary cultures, observe the cultures using a phase-contrast microscope. Some AdMSCs will be observed adhering to the surface of the flask. The medium should be changed twice a week.

[0115] Table 1 shows general methods for troubleshooting in protocols for isolating and culturing Ad MSCs.

[0116] [Table 1]

[0117] Protocol for Peripheral Blood Cell Isolation In one embodiment, the present invention discloses a protocol for the isolation of peripheral blood cells. 1. Inject the buffy coat into Part Ab with the nanoparticle-specific antibody reagent. Incubate at 37°C for 15-30 minutes with rocking. Place Part Ab on top of Part C for 2-5 minutes. Aspirate the supernatant from Part B, then add 10 mL of wash buffer to the Part Ab flask. Repeat this twice. Separate Part Ab from Part C. Add 10 mL of culture medium B+D to Part Ab and seal Part Ab with Part D. 2. Transfer the part Ab to a cell culture incubator at 37 °C, 5% CO for 5-7 days.

[0118] Protocol for converting Ad MSCs to HS / PCs: After initial seeding of primary Ad MSC cultures, once the cells reach approximately 90% confluence (7-13 days), Ad MSCs are passaged using the UL™ Hematopoietic Stem Cell (HS / PC) Conversion Kit (UL™ HS / PC-C Kit) disclosed in Example 2.

[0119] This protocol involves culturing actively growing cells at near-confluence in a single 60 mm culture dish or 25 cm 2Designed for subculturing in culture flasks. If using a different size culture vessel, adjust the reagent volumes accordingly.

[0120] In one embodiment, the reagents should not be pre-warmed before use. 1. Prepare UL™ HS / PC-C complete medium. Dissolve Reagent B (nutrient supplements) and C (conversion supplements) on ice. Once the reagents are completely dissolved, carefully pipette the reagents into the Reagent A bottle and mix well. 2. Remove all medium from the flask. 3. Wash the cells with 5 mL of Ca++ and Mg++ free PBS and place in the flask. Rock the flask to ensure the entire surface is covered. 4. Immediately remove the PBS solution from the flask and add 5 mL (for a 60 mm culture dish), 8 mL (for a 25 cm 2 Add UL™ HS / PC-C complete medium to the culture flask. 5. Incubate the flask in a 37°C incubator with 5% CO2 and humidity for 3-6 days. Change the medium approximately every 3 days.

[0121] Protocol for subculture of HS / PC after conversion. 1. Decant the medium from the cell culture dish or flask. Rinse the cells with 5 mL of calcium-free, magnesium-free Dulbecco's phosphate-buffered saline (D-PBS). Decant the D-PBS. 2. Add 1 mL of pre-warmed trypsin-EDTA (0.25%) to a 60 mm culture dish or 75 cm 2 Add 2 mL to the flask. 3. Incubate at 37°C until cells detach (observe every 2 minutes). Add 4.2 mL to 4 mL of cell culture growth medium and transfer the cell suspension into two or more new 100 mm culture dishes or 75 cm flasks. 5. After 48 hours of incubation, cells are continued to be cultured for 4 days in UL™ HS / PC-C complete medium without SB431542, CHIR99021, and Y-27632. 6. Subculture the converted HS / PCs or cryopreserve the converted HS / PCs.

[0122] In one embodiment, if the converted HS / PCs are not needed for further study, pipette the cells into a new 15 mL tube. Centrifuge at 100 x g for 5-10 minutes. Discard the supernatant and suspend the cell pellet in the appropriate reagent.

[0123] Protocol for cryopreservation of converted HS / PC. If surplus cells are available from subculture, they should be treated with an appropriate protective agent (eg, DMSO or glycerol) and stored at temperatures below -130°C (cryopreservation) until needed.

[0124] Cultures are observed under a microscope to determine the state of the culture (i.e., confluence, mitotic activity). In one embodiment, the present invention cryopreserves converted HS / PCs when the cultures are approximately 90% confluent and actively growing.

[0125] It is not recommended to warm the reagents before use.

[0126] Follow steps 1 to 4 for subculture of transformed HS / PC. 1. Resuspend the cell pellet in 2-3 mL of cold (4°C) UL™ Cryo Medium and transfer 1 mL (to obtain approximately 2-5 x 106 cells / mL) to a CryoFlex Tubing. 2. Calculate the number of viable cells per mL using a hemocytometer and dilute to the desired final cell density (5-10 x 10 viable cells / mL is recommended). 3. Cells are cryopreserved using a controlled rate freezer or other suitable device and then transferred to liquid nitrogen (vapor phase) for storage.

[0127] Results: After 24 hours of incubation with the conversion reagent, observe the cultures using a phase-contrast microscope. In one embodiment, some cells may detach into the medium in the flask, and some floating cells and debris may be present in the medium. After 72 hours, carefully change the medium to avoid dislodging loosely adherent cells. The converted cells continue to grow until they reach 85%-90% confluence.

[0128] Table 2 shows a troubleshooting chart for cryopreservation of converted HS / PC.

[0129] [Table 2]

[0130] Protocol for in vitro differentiation of converted HS / PCs into RBCs and platelets. This protocol involves culturing actively growing cells at near-confluence in a single 60 mm culture dish or 25 cm 2 Designed for subculturing in culture flasks. If using a different size culture vessel, adjust the reagent volumes accordingly.

[0131] It is not recommended to change the medium between days 10 and 25. In one embodiment, it is recommended to add fresh medium twice a week.

[0132] Prepare UL™-RBC / Platelet Complete Medium: Dissolve Reagent B (nutritional supplement) and Reagent C (RBC and platelet supplement) on ice. Once the reagents are completely dissolved, carefully pipette the reagent into the Reagent A bottle and mix well. 1. After transformation, place the HS / PC in a 60 mm culture dish or a 25 cm 2 After initial seeding in the flask, once the cells reach approximately 90% confluence (7-13 days), remove all culture medium from the culture dish or flask. Then, add 5 mL (60 mm culture dish) or 10 mL (25 cm) of medium to the culture dish or flask. 2Add complete red blood cell and platelet medium (UL™-h or m RBC kit) to the flask. Add 2.8 mL of RBC / platelet complete medium to the cell culture dish or flask twice a week. 3. On days 18–25, collect the supernatant containing floating cells into a new 50 mL centrifuge tube. 4. Spin down the cells at 1000 rpm for 10 minutes and wash the cells two or three times with 10 mL of Ca++ and Mg++ free PBS. 5. Save the cell pellet for future use.

[0133] Results: On days 18-21 of culture, few small, dark, round cells were observed floating in the supernatant.

[0134] Table 3 shows troubleshooting strategies for in vitro differentiation of converted HS / PCs into RBCs and platelets.

[0135] [Table 3]

[0136] Protocol for in vitro differentiation of converted HS / PCs into WBCs This protocol involves culturing actively growing cells at near-confluence in a single 60 mm culture dish or 25 cm 2 Designed for subculturing in culture flasks. If using a different size culture vessel, adjust the reagent volumes accordingly.

[0137] It is not recommended to change the medium between days 10 and 35. In one embodiment, it is recommended to add fresh medium twice a week.

[0138] Prepare UL™ WBC Complete Medium. Dissolve Reagent B (basal nutritional supplement) and Reagent C (WBC supplement) on ice. Once the reagents are completely dissolved, carefully pipette Reagent B and Reagent C into Reagent A bottle and mix well. 1. After transformation, place the HS / PC in a 60 mm culture dish or a 25 cm 2 After initial seeding in flasks, once the cells reach approximately 90% confluence (7-13 days), remove all culture medium from the dish or flask. Then, add 5 mL (for 60 mm dishes) or 10 mL (for 25 cm flasks) of culture medium. 2 Add complete leukocyte medium (UL™ h or m WBC kit) to the flask. Add 2.8 mL of WBC complete medium to the cell culture dish or flask twice a week. 3. On days 20 to 35, collect the supernatant containing floating cells into a new 50 mL centrifuge tube. 4. Spin down the cells at 1000 rpm for 10 minutes and wash the cells two or three times with 10 mL of Ca++ and Mg++ free PBS. 5. Save the cell pellet for future use.

[0139] Results: From day 10 to day 35 of culture, few small, dark, round cells were observed floating in the supernatant.

[0140] Table 4 shows troubleshooting strategies for in vitro differentiation of converted HS / PCs into WBCs.

[0141] [Table 4]

[0142] A protocol to improve the efficiency of in vitro gene transfection using transformed HS / PC as gene carrier cells. This protocol is designed for subculturing near-confluent, actively growing cells in a single 60 mm culture dish. If using a different size culture vessel, adjust the volumes of reagents accordingly.

[0143] It is not recommended to use the UL™ Gene Transfection Enhancer Kit (UL™ h or m TFK) after 24 hours of transfection. 1. Prepare UL™ Gene Transfection Enhancer Kit (UL™ h or m TFK) complete medium. Thaw Reagent B (nutrient supplement) and Reagent C (gene transfection enhancer supplement) from the UL™ Human or Mouse Gene Transfection Enhancer Kit (UL™ h or m TFK) on ice. Once the reagents are completely dissolved, carefully pipette the reagents into the Reagent A bottle and mix well. 2. After 24 hours of normal transfection, remove all culture medium from the dish and then add 5 mL (60 mm dish) of UL™ Gene Transfection Enhancer Complete Medium (UL™ h or m TFK Kit) to the culture dish. 3. Change the TFK medium twice a week. When the cells reach confluence, harvest them and select positive cells. 4. Culture the pure positive cells in TFK complete medium for another 3-6 days, then replace the culture medium with cell culture basal medium A supplemented with nutritional supplement B. Replace the medium twice a week until the cells have proliferated to a sufficient number for gene therapy.

[0144] Results: Six days after transfection there should be more than 70% positive cells (if a visible marker is present in the line).

[0145] Table 5 shows troubleshooting steps for improving the in vitro gene transfection efficiency using transformed HS / PCs as gene carrier cells.

[0146] [Table 5]

[0147] Example 3 - Reagents and kits for tissue single cell isolation and Ad MSC culture In one embodiment, the enzymatic digestion reagent (UL™ Tissue MSC Isolation—UL™ TMSC-IS Kit) contains 1×PBS supplemented with 0.25% collagenase type I / 5 mM glucose / 1.5% BSA.

[0148] In one embodiment, the nanoparticle-specific antibody reagent comprises lipid-based nanoparticles (LNPs) or polymer-based nanoparticles (PNPs) of less than 100 nm, depending on the user's goals, along with a specific antibody.

[0149] 1. UL™ Mesenchymal Stem Cell Expansion Medium (UL™-MSC-E) In one embodiment, the present invention discloses a cell culture reagent for Ad MSC and blood cell culture UL™ Mesenchymal Stem Cell Expansion Medium (UL™-MSC-E).

[0150] In one embodiment, UL™ Mesenchymal Stem Cell Expansion Medium (UL™-MSC-E) comprises one or more of the following four components: A. Ad MSC (adherent cell) culture basal medium (stored at 4°C): 500 mL of high-glucose DMEM / F12 supplemented with L-glutamine, 2.438 g / L sodium bicarbonate, penicillin-streptomycin (10,000 U / mL), 1% NEAA, and 1% HEPES. B. Basal Nutrient Supplement (store at -20°C): 25 mL fetal bovine serum (10%) and 25 mL horse serum (10%) (both sera require heat inactivation). C. Cell suspension culture basal medium (store at 4°C): 500 mL of high glucose RPMI 1640 supplemented with L-glutamine and 2.438 g / L sodium bicarbonate, penicillin-streptomycin (10,000 U / mL), 1% NEAA, and 1% HEPES. D. Nutritional supplement (store at -20°C): 50 mL fetal bovine serum (10%) (heat inactivated).

[0151] 2. UL™ Human or Mouse HS / PC Conversion Kit (UL™-hHS / PC-C Kit) In one embodiment, the present invention discloses a reagent for converting Ad MSCs to HS / PCs in vitro.

[0152] The UL™ human or mouse hematopoietic stem cell (HS / PC) conversion kit (UL™-hHS / PC-C kit) contains one or more of the following three components: A. Cell culture basal medium (store at 4°C): 500 mL of high-glucose DMEM / F12 supplemented with L-glutamine and 2.438 g / L sodium bicarbonate, penicillin-streptomycin (10,000 U / mL), 1% NEAA, and 1% HEPES. B. Basal Nutrient Supplement (store at -20°C): 25 mL fetal bovine serum (10%) and 25 mL horse serum (10%) (both sera require heat inactivation). C. Conversion supplements from Table 6 (store at -80°C).

[0153] [Table 6]

[0154] 3. UL™ Human or Mouse Red Blood Cell and Platelet Kit (UL™ h or m RBC Kit) for Differentiation of Post-Conversion HS / PCs into RBCs and Platelets In one embodiment, the present invention discloses reagents for inducing differentiation of converted HS / PCs into RBCs and platelets in vitro and in vivo.

[0155] UL™ Human or Mouse Red Blood Cell and Platelet Kits contain one or more of the following three components: A. Cell culture basal medium (store at 4°C): 500 mL of high-glucose DMEM / F12 supplemented with L-glutamine and 2.438 g / L sodium bicarbonate, penicillin-streptomycin (10,000 U / mL), 1% NEAA, and 1% HEPES. B. Basal Nutrient Supplement (store at -20°C): 50 mL fetal bovine serum (10%) and 50 mL horse serum (10%) (both sera require heat inactivation). C. RBC and platelet supplements listed in Table 7 (-80 o (Save as C).

[0156] [Table 7]

[0157] 4. UL™ Human or Mouse Leukocyte Kit (UL™ h or m WBC Kit) In one embodiment, the present invention discloses reagents for in vitro and in vivo differentiation of peripheral WBCs from converted HS / PCs into various lineages. The UL™ human or mouse leukocyte kit (UL™ h or m WBC kit) contains one or more of the following three components: A. Cell culture basal medium (store at 4°C): 500 mL of high-glucose DMEM / F12 supplemented with L-glutamine and 2.438 g / L sodium bicarbonate, penicillin-streptomycin (10,000 U / mL), 1% NEAA, and 1% HEPES. B. Basal Nutrient Supplement (store at -20°C): 50 mL fetal bovine serum (10%) and 50 mL horse serum (10%) (both sera require heat inactivation). C. WBC supplement from Table 8 (store at -80°C).

[0158] [Table 8]

[0159] 5. UL™ Human or Mouse Gene Transfection Enhancer Kit (UL™ h or m TFK) In one embodiment, the present invention discloses a system for enhancing gene transfer of converted HS / PCs in vitro. The UL™ human or mouse Gene Transfection Enhancer Kit (UL™ h or m TFK) contains one or more of the following three components: A. Cell culture basal medium (store at 4°C): 500 mL of high-glucose DMEM / F12 supplemented with L-glutamine and 2.438 g / L sodium bicarbonate, penicillin-streptomycin (10,000 U / mL), 1% NEAA, and 1% HEPES. B. Basal Nutrient Supplement (store at -20°C): 50 mL fetal bovine serum (10%) and 50 mL horse serum (10%) (both sera require heat inactivation). C. Gene transfection enhancer supplements from Table 9 (store at -80°C).

[0160] [Table 9]

[0161] Example 4 - Results of isolation, culture and conversion of Ad MSCs into HS / PCs Identification of adipose-derived mesenchymal stem cells (Ad-MSCs) isolation from both human and mouse adipose tissue using a modified one-step method. Figure 1 shows that both human and mouse adipose-derived mesenchymal stem cells (Ad-MSCs) stained positively with Oil Red O. Oil Red O staining is a common method for identifying adipocytes.

[0162] 2 and 3 show the growth curves of Ad-MSC cultures. The proliferation of Ad-MSCs increased linearly after 7 days of culture.

[0163] Figures 4 to 6 show the results of imaging flow cytometry. These results show that the Ad-MSC-specific markers CD73, CD90, CD90.2, and CD105 are positively expressed in both human and mouse adipose-derived mesenchymal stem cells (Ad-MSCs). However, while the CD90.2 marker was expressed in mouse bone marrow, CD73 and CD105 were not. Therefore, the cells are adipose-derived mesenchymal stem cells (Ad-MSCs) isolated by a one-step isolation approach. The one-step isolation and Ad-MSC culture medium system can be used in the art.

[0164] Conversion of primary Ad MSCs to hematopoietic stem / progenitor cells (HS / PC) in vitro using UL™-h or m HS / PC-C medium. Figures 7 and 8 show the conversion of Ad MSCs to HS / PCs using UL™ h or m HS / PC-C complete medium for culture. After conversion, HS / PCs began to form clones on day 3, and the clones became more distinct and increased in size with the extension of culture time. These results were similar for both human and mouse cells. No clones were observed in Ad MSCs cultured in UL™ HS / PC-C complete medium.

[0165] Figure 9 shows converted mouse HS / PCs stained with FITC-Sca-1 (pink) marker (Sca-1 is a mouse hematopoietic stem cell-specific marker) and sorted by flow cytometry. FITC-Sca-1 positive cells accounted for 11.7% of all sorted cells. This indicates a conversion rate of 11.7% for HS / PCs, which is significantly higher than that of existing technologies.

[0166] Figures 10-11 show that the markers CD90.2 (blue), c-kit (CD117) (purple), CD48 (green), CD105 (red), and CD150 (pink) (murine hematopoietic stem cell-specific markers) are positively expressed in Sca-1-positive murine HS / PCs after conversion (D). As shown in Figure 11, the FITC-Sca-1 marker (green) shows positive expression in murine HS / PCs cultured as live cells and converted (E).

[0167] FIG. 12 shows flow cytometry results reflecting the positive expression of CD34 (green, red), CD90 (yellow, red), and CD49f (pink) markers in human cell-derived converted HS / PCs.

[0168] 7 to 12, both human and mouse Ad MSCs can form clones in vitro in UL™ HS / PC-C medium without the introduction of any exogenous genes and can have positive expression of hematopoietic stem cell-specific markers. However, Ad MSCs cultured in UL™ HS / PC-C medium do not form clones.

[0169] A system for differentiation of post-conversion HS / PCs into various peripheral blood cell lineages both in vitro and in vivo Expression of glycophorin A (CD235a in humans and TER 119 in mice) and fetal hemoglobin markers upon differentiation of post-conversion HS / PCs.

[0170] In one embodiment, Figures 13-19 show in vitro and in vivo results.

[0171] Figure 13 shows that after 18-23 days of culture in UL™ hRBC medium, supernatant cells from human converted HS / PCs positively expressed the glycophorin A (CD235a) (green) marker. The top panel of Figure 13 shows a control of human blood RBCs stained with CD235a (green).

[0172] Figure 14 shows imaging flow cytometry results confirming the conversion of human HS / PCs after 18-23 days of culture in UL™ hRBC complete medium. In the bottom panel of Figure 14, new floating cells are present in the culture medium, positively expressing the specific markers glycophorin A (CD235a) (yellow) and fetal hemoglobin (Hb) (green). The top panel of Figure B shows a positive control of human blood RBCs stained for CD235a and fetal hemoglobin (Hb). Because fetal hemoglobin (Hb) is the major oxygen-carrying protein in the fetus, only the CD235a (yellow) marker is expressed on human blood RBCs, and no fetal hemoglobin (green) marker is expressed on human RBCs.

[0173] Figure 15 shows that after 18-23 days of culture in UL™ mRBC medium, supernatant cells from murine converted HS / PCs positively expressed the glycophorin A (TER 119) (green) marker. TER 119 is a murine erythrocyte-specific marker.

[0174] Figure 16 shows imaging flow cytometry results confirming the presence of converted mouse HS / PCs after 18-23 days of culture in UL™ mRBC complete medium. In the bottom panel of Figure 16, new floating cells are present in the culture medium, and the floating cells positively express the specific markers TER 119 (yellow) and fetal Hb (green). In the top panel of Figure 16, a positive control of mouse blood RBCs stained with TER 119 (yellow) and fetal Hb (green) is shown. Because fetal Hb is the primary oxygen-carrying protein in the fetus, fetal Hb (green) marker expression is absent in mouse blood RBCs.

[0175] Figures 17 and 18 show the results of imaging flow cytometry, confirming that CD45.1 (red), TER119 (green), and CD41 markers were positively expressed in CD45.2 mouse blood cells transplanted with HS / PCs after conversion of CD45.1 mice on day 23. CD41 is a specific marker for mouse platelets.

[0176] Figure 19 shows the transplantation of HS / PCs from CD45.1 mice into six CD45.2 mice, four of which survived healthy for 18 months.

[0177] Therefore, we can conclude that (1) glycophorin A (CD235a) (TER 119) and fetal hemoglobin (Hb) markers were positively expressed in cells in the culture supernatant of human and mouse converted HS / PCs after 18 to 23 days of in vitro culture in UL™ h or m RBC medium; (2) glycophorin A (TER 119), CD41, and CD45.1 markers were positively expressed in the blood of CD45.2 mice transplanted with converted HS / PCs from CD45.1 mice on day 23; (3) converted hematopoietic stem cells (HS / PCs) differentiated in vitro and in vivo into cells positively expressing fetal hemoglobin, glycophorin A, and CD41 markers in UL™ h or m RBC medium; and (4) as a result of transplantation, converted HS / PCs were demonstrated to be able to reconstitute the immune system in vivo and are safe for reinfusion into the body.

[0178] This conclusion further confirms that HS / PCs converted from AdMSCs can differentiate in vitro and in vivo into blood cells with positive expression of fetal Hb, glycophorin A, and CD41 markers under the innovative UL™ h or m RBC system without the insertion of any exogenous genes.

[0179] Differentiation of HS / PCs into leukocyte marker-positive cells after conversion in vitro and in vivo using UL™ human or mouse leukocyte kits (UL™ h or m WBC kits) In one embodiment, Figures 20-22 show in vitro and in vivo results.

[0180] Figure 20 shows the CBC results reflecting cells in the supernatant of murine post-conversion HS / PCs cultured for 25 days in UL™ mWBC medium. WBC was 11.2 x 10 3 cells / μL, and LYM (lymphocytes) is 2.3 × 10 3 cells / μL, and monocytes are 0.8 × 10 3 cells / μL, and GRAN (granulocytes) is 8.1 × 103 Counts / μL.

[0181] Figure 21 reflects the CBC results showing cells in the supernatant of mouse post-conversion HS / PCs cultured for 25 days under normal medium. WBC was 0.9 x 10 3 cells / μL, and LYM (lymphocytes) is 0.6 × 10 3 cells / μL, and monocytes are 0.1 × 10 3 cells / μL, and GRAN (granulocytes) is 0.2 × 10 3 Count / μL (control)

[0182] Figure 22 shows flow cytometry analysis showing that CD45.1 mouse leukocyte markers (CD45.1, CD3 / CD45.1, CD19 / CD45.1, CD25 / CD45.1, CD11b / CD45.1, and CD68 / CD45.1) were positively expressed in the peripheral blood of CD45.2 mice transplanted with HS / PCs after conversion of CD45.1 mice on day 31.

[0183] Therefore, this analysis further confirms that HS / PCs converted from Ad MSCs can differentiate into blood cells with positive expression of multilineage leukocyte markers in vitro and in vivo under our innovative UL™ h or m WBC medium without the insertion of any exogenous genes.

[0184] Enhancement system (UL™ TFK) for gene transfer of HS / PC after in vitro transformation. FIG. 23 shows that in transformed HS / PCs transfected with pLenti-GFP, there are at least 25 GFP-positive cells (green) after 3 days of culture in UL™ TFK medium.

[0185] FIG. 24 shows that in transformed HS / PCs transfected with pLenti-GFP medium, only one GFP-positive cell was present on day 3 of culture under normal medium conditions.

[0186] Comparing the gene transfer efficiency of UL™ TFK medium and regular medium, the transfection efficiency of UL™ TFK medium was approximately 25 times higher than that of regular medium.

[0187] Therefore, the converted HS / PCs derived from Ad MSCs can be effective carrier cells for gene transfer, and the converted HS / PCs can be selectively differentiated into desired cells (e.g., T cells, B cells, NK cells, etc.).

[0188] Furthermore, UL™ TFK can improve gene transfection efficiency. The combination of converted HS / PC and UL™ TFK solves some of the major problems facing current gene therapy, specifically the challenges of culturing and transfecting gene carrier cells under in vitro conditions.

[0189] Example 5 - Protocol for isolation, expansion and conversion of Ad MSCs into HS / PCs, and differentiation of converted HS / PCs into various blood cells Methods and Procedures: Adipose tissue from a total of 10 mice from five strains, and three human Ad MSC cell lines.

[0190] In one embodiment, the present invention discloses an in vitro method.

[0191] Isolation and expansion of Ad-MSCs: In one embodiment, the present invention discloses an automated process for the isolation and expansion of Ad MSCs. 1. Wash 0.5 g of mouse intraperitoneal adipose tissue three times with washing buffer and cut it into 0.5 mm pieces with scissors under a sterile hood. 2 Add 2 mL of wash buffer to the wash dish and transfer the whole to the grinder. 2. UL-Select a program on the automated multifunctional single cell processing device and press the start button. 3. Transfer the cell flasks from the automated, multi-functional single-cell processing device to a cell culture incubator at 37°C, 5% CO2, and humidified conditions. Change the medium twice a week. No cells should be visible in the culture dishes for the first 2 or 3 days.

[0192] In one embodiment, the present invention discloses a manual process for the isolation and expansion of Ad MSCs. Wash 1.0.5 g of mouse intraperitoneal adipose tissue three times with washing buffer and cut it into 0.1-3 mm pieces with scissors under a sterile hood. 2 Add 2 mL of wash buffer to the wash dish and then transfer the entire dish to a 15 cm sterile centrifuge tube containing 8 mL of enzymatic digestion reagent (UL™ TMSC-IS kit). Incubate at 37°C for 45-60 minutes with shaking. 2. When the liquid becomes cloudy (indicating complete breakdown of the adipose tissue), transfer all of the liquid to a new 50 mL sterile centrifuge tube equipped with a 70 mm cell strainer. Then, pour the liquid into the 100 mm cell strainer of a 100 mm culture dish and rinse the filter membrane with 10 mL of washing solution. Tilt the culture dish and use a glass pipette to vacuum-suction the liquid from the bottom of the dish (trying to make the top of the liquid surface touch the bottom of the dish). Incubate the culture dish at room temperature for 10 minutes, then gently add washing solution. Aspirate the washing solution (the lotion) as described above and repeat this process twice. 3. 10 mL of UL™ Ad MSC Expansion Medium (UL™ Ad MSC-E) is added to the culture dish, and the cell dish is cultured in an incubator at 37°C, 5% CO2, and humidified conditions. The medium is changed twice a week. No cells will be visible in the culture dish for the first 2 or 3 days.

[0193] In vitro conversion of Ad MSCs to HS / PCs: 1. Once Ad MSCs have grown to approximately 90% confluence, wash the cells with 1x PBS. 2. Add 5 mL of UL™ HS / PC-C complete medium to a 60 mm plate or 8 mL to a 25 cm flask and culture for 48 hours. 3. Replace UL™ HS / PC-C complete medium with UL™ HS / PC-C Medium-2 and continue culturing. Change medium twice a week.

[0194] Typically, HS / PCs can be converted in approximately 6 to 8 days. Cells are identified (indented) by flow cytometry analysis. 4. If the clone grows, subculture it according to the manual.

[0195] After conversion, the cells are differentiated in vitro into RBC and platelet marker-positive cells. 1. After conversion, once the HS / PCs have grown to approximately 90% confluence, wash the cells with 1x PBS. 2. Add 5 mL for 60 mm plates or 8 mL for 25 cm flasks of UL™ RBC complete medium and culture the cells in a humidified incubator at 37° C., 5% CO 2 .

[0196] Medium should be added twice a week for the first 10-15 days. 3. Similarly, add 5 mL of UL™ RBC complete medium to a 60 mm plate or 8 mL to a 25 cm flask and culture the cells in a humidified incubator at 37°C, 5% CO2. 4. Similarly, from day 11 to day 23, add 10 mL or 20 mL of UL™ RBC Complete Medium twice a week. Be careful not to discard the old medium and carefully observe cell growth. If new small dark cells are found in the culture medium, collect them for further study. At the same time, add new medium to the culture dish for further cultivation.

[0197] After the alteration, the cells were converted to WBC marker-positive cells in vitro. 1. After conversion, once the HS / PCs have grown to approximately 90% confluence, wash the cells with 1x PBS. 2. Add 5 mL of UL™ WBC complete medium to a 60 mm plate or 8 mL to a 25 cm flask and culture the cells in a humidified incubator at 37°C, 5% CO2.

[0198] Medium should be added twice a week for the first 15-38 days. 3. Similarly, add 5 mL of UL™ WBC complete medium to a 60 mm plate or 8 mL to a 25 cm flask and culture the cells in a humidified incubator at 37°C, 5% CO2. 4. Similarly, from day 16 onwards, add 10 mL or 20 mL of UL™ RBC complete medium twice a week. Be careful not to discard the old medium and carefully observe cell growth. If new small dark cells appear in the culture medium, collect them for further study. At the same time, add new medium to the culture dish for further cultivation.

[0199] Enhancement system for gene transfer of HS / PC after in vitro transformation. 1. Isolate and culture Ad MSCs using the UL™ Ad MSC-IS kit and the UL™ Ad MSC-E kit. 2. Ad MSCs are converted into HS / PCs with UL™ HS / PC-C, the cells are cultured for 6 to 8 days, and then the converted HS / PCs are subcultured at 70% or 80% confluence. 3. Perform transfection experiments using any transfection method. 4. After overnight transfection of cells, replace with 5 mL or 10 mL of UL™ TFK complete medium. Continue culturing at 37°C, 5% CO2, and humidified conditions. 5. Target cells can be selected by any method, and positive cells can be selected for future purposes. Furthermore, transfection of transformed HS / PCs can also selectively differentiate them into desired cells (e.g., T cells, B cells, NK cells, etc.). This can prevent patients from losing treatment opportunities due to cell shortages. 6. UL™ Ad MSC-E Kit can increase the chances of cell expansion and subculture. Please follow the manual for details.

[0200] In vitro procedure: 1. The isolation and culture of Ad MSCs from CD45.1 mice is disclosed above. 2. Conversion of Ad MSCs to HS / PCs for CD45.1 mice is disclosed below. 3. After sorting the positively converted HS / PCs, the cells are cultured in UL™ Ad MSC-E medium. 4. Once sufficient marker-positive HS / PCs have proliferated, harvest the cells and wash them 3-4 times with 1x D-PBS to thoroughly remove protein components. 5. On the day of transplantation, isolate CD45.2 mouse bone marrow cells. 6. 1-2 x 10 in 0.1 mL of saline 7 Suspend 1-2 x 10 converted CD45.1-HS / PCs in 0.1 mL of saline. 7 CD45.2 BM cells are then suspended in 100 mL of PBS. Both cells are then mixed thoroughly and kept on ice before injection into mice. 7. CD45.2 recipient mice require two rounds of irradiation: the first round 4 hours before injecting cells into the CD45.2 recipients, and the second round 1 hour before injection. 8. The mixed cells are injected into the facial muscles of recipient mice. 9. Keep CD45.2 recipient mice healthy and closely monitored. 10. Bleed mice via the orbit or tail vein to begin monitoring approximately 3 weeks after implantation. 11. Analyze by flow cytometry.

[0201] analysis: 1. Oil Red O staining: performed according to general knowledge in the art. 2. Flow cytometry analysis: Follow the manufacturer's manual for details on cell labeling.

[0202] Ad MSC Panel: In humans: FITC-CD90, PE-CD73, APC-CD105 For mice: FITC-CD90.2, PE-CD73, APC-105

[0203] B. HSC panel: In humans: Brilliant Violet 421™ anti-human / mouse CD49f Brilliant Violet 605™ anti-mouse CD45 antibody Alexa Fluor® 647 anti-human CD34 antibody FITC anti-human CD48 antibody PE / Cy5 anti-human CD135 (Flt-3 / Flk-2) PE anti-human CD90 (Thy1) antibody APC / Cy7 anti-human CD10 antibody

[0204] With a mouse: Brilliant Violet 421™ anti-human / mouse CD49f Brilliant Violet 711™ anti-mouse Ly-6A / E (Sca-1) antibody PE anti-mouse CD90.2 antibody PE / Cy7 anti-mouse CD150 (SLAM) antibody PE / Cy5 anti-mouse CD135 antibody Brilliant Violet 605™ anti-mouse CD45 antibody Alexa Fluor® 647 anti-mouse CD34 antibody FITC anti-mouse CD48 antibody APC / Cy7 anti-mouse CD117 (cKit) antibody

[0205] C. RBC Panel: In humans: FITC anti-human CD235a PE anti-human CD62P (P-selectin) antibody APC anti-human CD42b antibody

[0206] With a mouse: FITC anti-mouse TER-119 / erythroid cells PE anti-mouse / rat CD62P (P-selectin) antibody APC anti-mouse CD41 antibody

[0207] D. WBC Panel: In humans: Brilliant Violet 421™ anti-human CD25 antibody Brilliant Violet 510™ anti-human CD19 antibody Brilliant Violet 711™ anti-human CD14 antibody Anti-human CD3 PE-Cy5 / CD4 PE / CD8 FITC cocktail PE / Cy7 anti-human CD1c antibody APC anti-human CD56 (NCAM) antibody APC / Cy7 anti-human CD68 antibody

[0208] With a mouse: Brilliant Violet 421™ anti-mouse CD25 antibody Brilliant Violet 510™ anti-mouse CD19 antibody Brilliant Violet 711™ anti-mouse CD115 (CSF-1R) PE anti-mouse CD3 antibody PE / Cy7 anti-mouse CD11c antibody APC anti-mouse CD49b (pan-NK cell) antibody APC / Cy7 anti-mouse CD68 antibody

[0209] The foregoing description of exemplary embodiments of the present invention has been presented for purposes of explanation and illustration. The precise forms disclosed above are not intended to be exhaustive or to limit the invention, and modifications and variations are possible based on the foregoing disclosure or may be acquired from the practice of the present invention. The embodiments were chosen and described to explain the principles of the invention and its practical application to others skilled in the art and to enable them to utilize the invention in various forms and modifications for their intended purposes. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims

1. 1. A method for producing hematopoietic stem / progenitor cells (HS / PC) and blood cells from adipose-derived mesenchymal stem cells (Ad MSC), comprising: obtaining biological cells of interest; expanding the biological cells; Obtaining a pure Ad MSC cell line from the expanded living cells; converting the pure Ad MSC cell line into HS / PC in vitro; Expanding the converted HS / PC in vitro; and Obtaining the expanded HS / PCs; A method comprising:

2. further comprising differentiating the expanded HS / PCs into blood cell lines; 10. The method of claim 1, wherein the blood cell lines comprise at least one of white blood cells (WBCs), red blood cells (RBCs), and platelets.

3. The method of claim 1 , wherein the step of converting the pure Ad MSC cell line into HS / PC does not use any exogenous genes.

4. obtaining the pure Ad MSC cell line, isolating the pure Ad MSC cell line from the biological sample; and Culturing the pure Ad MSC cell line; The method of claim 1 , comprising:

5. converting the pure Ad MSC cell line into the HS / PC; The method of claim 1 , comprising using an HS / PC conversion kit.

6. The HS / PC conversion kit comprises: Ad MSC basal medium, Basic nutritional supplements, and Base conversion aid mixture The method of claim 5 , comprising:

7. converting the pure Ad MSC cell line into the HS / PC; mixing the mixture of the basal nutritional supplements and the basal conversion supplements into the Ad MSC basal medium to prepare a HS / PC-C complete medium; adding the HS / PC-C complete medium to the obtained pure Ad MSC cell line; and Incubating the pure Ad MSC cell line in the HS / PC-C complete medium for a period of time; The method of claim 6, comprising:

8. 6. The method of claim 5, wherein the conversion of the pure Ad MSC cell line to the HS / PC has a conversion efficiency of greater than 10%.

9. The method of claim 5, wherein the converted HS / PC has a gene transfection efficiency 25 times higher than that of normal converted HS / PC that has not been converted with the HS / PC conversion kit.

10. The method of claim 5 , wherein the transformed HS / PC comprises gene carrier cells.

11. 1. A kit for converting adipose-derived mesenchymal stem cells (Ad MSCs) into hematopoietic stem / progenitor cells (HS / PCs), comprising: Ad MSC basal medium, Basic nutritional supplements, and Base conversion aid mixture Including, A HS / PC conversion kit for converting the Ad MSCs into HS / PCs.

12. 12. The kit of claim 11, wherein the basal nutritional supplement comprises fetal bovine serum and horse serum.

13. The kit of claim 11, wherein the basal conversion supplement mixture comprises at least one of insulin, holotransferrin, sodium selenite (ITS solution), L-ascorbic acid, GM-CSF, SCF, VEGF, IGF-I, IGF-II, IL-3, Flt3-L, thrombopoietin (TPO), dexamethasone, fatty acid-free BSA, 1-thioglycerol, SB431542, CHIR99021, and Y-27632.

14. The kit of claim 14, wherein the basal conversion supplement mixture comprises insulin, holotransferrin, sodium selenite (ITS solution), L-ascorbic acid, GM-CSF, SCF, VEGF, IGF-I, IGF-II, IL-3, Flt3-L, thrombopoietin (TPO), dexamethasone, fatty acid-free BSA, 1-thioglycerol, SB431542, CHIR99021, and Y-27632.

15. 1. A red blood cell (RBC) and platelet differentiation kit for differentiating hematopoietic stem / progenitor cells (HS / PC) into RBCs and platelets, comprising: basal cell culture medium, Basic nutritional supplements, and RBC supplement mixture Including, An RBC and platelet differentiation kit for differentiating the HS / PC into at least one of RBC and platelet.

16. 16. The kit of claim 15, wherein the basal nutritional supplement comprises fetal bovine serum and horse serum.

17. 16. The kit of claim 15, wherein the RBC supplement mixture comprises at least one of insulin, holotransferrin, sodium selenite (ITS solution), L-ascorbic acid, SCF, VEGF, IGF-I, IGF-II, IL-3, Flt3-L, thrombopoietin (TPO), dexamethasone, fatty acid-free BSA, 1-thioglycerol, EPO, ferrous sulfate, PDGF BB, and activin A.

18. A WBC differentiation kit for differentiating hematopoietic stem / progenitor cells (HS / PC) into white blood cells (WBC), comprising: basal cell culture medium, Basic nutritional supplements, and WBC Adjuvant Mixture Including, A WBC differentiation kit for differentiating the HS / PC into WBC.

19. 20. The kit of claim 18, wherein the basal nutritional supplement comprises fetal bovine serum and horse serum.

20. 19. The kit of claim 18, wherein the basal conversion supplement mixture comprises at least one of insulin, holotransferrin, sodium selenite (ITS solution), L-ascorbic acid, SCF, VEGF, IGF-I, IGF-II, IL-3, Flt3-L, thrombopoietin (TPO), dexamethasone, fatty acid-free BSA, 1-thioglycerol, BMP4, IL-7, IL-12, IL-11, IL-6, IL-2, FGF-b, TGF-a, TGF-b1, TGF-b2, TGF-b3, IL-1a, IL-1b, IL-4, IL-5, IL-8, IL-10, IL-12, IL-32a, mIL-36R2, and GM-CSF.

21. An automated and multi-functional single cell processing and culture device, comprising: housing, at least one small fluid volume dispenser system disposed within the housing; and a tissue disruption system disposed within the housing below the small fluid volume dispenser system; It is equipped with A device wherein the small fluid volume dispenser system is configured to dispense at least one fluid, and the tissue disruption system receives the at least one fluid.

22. 22. The device of claim 21, wherein the small fluid volume dispenser system is configured to dispense a volume of the at least one fluid between about 5 mL and 10 mL.

23. 22. The device of claim 21, wherein the small fluid volume dispenser system comprises a cylinder containing one or more pre-filled cartridges.

24. 24. The device of claim 23, wherein the small fluid volume dispenser system comprises a top needle positioned above the one or more pre-filled cartridges and a bottom needle positioned below the one or more pre-filled cartridges.

25. 25. The device of claim 24, wherein the upper needle and the lower needle are configured to move vertically to pierce the filled cartridge.

26. 22. The device of claim 21, wherein the tissue disruption system comprises a vial mechanically connected to a motor.

27. 27. The device of claim 26, wherein the vial contains a top grinder plate, a bottom grinder plate, and a filter disposed below the top grinder plate and the bottom grinder plate.

28. 27. The device of claim 26, wherein the top grinder plate and the bottom grinder plate are arranged to form a sample placement location for receiving a biological sample.

29. 30. The device of claim 28, wherein the tissue disruption system further comprises a tube fluidly connected at a first end to the bottom of the vial.

30. 30. The device of claim 29, wherein the tube is fluidly connected at a second end to a dispenser funnel.

31. 22. The device of claim 21, comprising a cell flask rotor, said cell flask rotor comprising a plurality of magnetic nanoparticles.

Images