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CN-121975722-A - Stem cell preparation for treating prostate diseases and preparation method and application thereof

CN121975722ACN 121975722 ACN121975722 ACN 121975722ACN-121975722-A

Abstract

The invention discloses a preparation method and application of a prostate lineage progenitor cell obtained by multi-stage induction of a human induced pluripotent stem cell. The method comprises the steps of sequentially inducing and forming definitive endoderm, hindgut genitourinary Dou Zu cells and prostate progenitor cells, wherein a synergistic regulation system of LDN193189 and CHIR99021 is simultaneously added in the third stage, so that the lineage specification efficiency and the cell expansion capacity are obviously improved. Further sorting is carried out by combining membrane markers of high expression of CD49f, positive Trop2, low expression of CD26 and negative CD117, so that the cell population with high purity, low heterogeneity and immunoregulatory potential can be obtained. The cells can reconstruct glandular structures and secrete prostate specific proteins in animal models while improving inflammatory microenvironment through immune tolerance-related pathways. The process can realize mass production, maintain the activity and stability of the product, and is suitable for treating chronic prostatitis, postoperative tissue defect, dysfunction and other diseases.

Inventors

  • WAN CHENG

Assignees

  • 广东沃博生物医药科技有限公司

Dates

Publication Date
20260505
Application Date
20260211

Claims (10)

  1. 1. A method for preparing human prostate progenitor cells, said method comprising the following steps, performed sequentially: Step one, adding an Activin A, a CHIR99021 and a PI3K inhibitor into a human induced pluripotent stem cell culture system to induce and form definitive endoderm cells; step two, adding FGF4, all-trans retinoic acid, CHIR99021 and BMP4 into the culture system of the cells obtained in the step one, and inducing to form hindgut progenitor cells; and thirdly, adding dihydrotestosterone, fibroblast growth factor 10, keratinocyte growth factor, TGF-beta receptor inhibitor A83-01, 0.1 mu M LDN193189 and 3 mu M CHIR99021 into the culture system of the cells obtained in the second step, and inducing to form the human prostate progenitor cells.
  2. 2. The method according to claim 1, wherein the culturing time in the third step is 10 days.
  3. 3. A population of human prostate progenitor cells prepared by the method of claim 1 or 2.
  4. 4. A population of human prostate progenitor cells according to claim 3, characterized in that the proportion of NKX3-1 protein positive cells in the population of human prostate progenitor cells is not less than 70%, preferably not less than 73%.
  5. 5. The population of human prostate progenitor cells according to claim 3 or 4, wherein the population of human prostate progenitor cells has a high expression of CD49f protein and a positive expression of Trop2 protein has a cell fraction of not less than 68%.
  6. 6. The population of human prostate progenitor cells according to any one of claims 3 to 5, wherein not less than 90% of the cells in the population of human prostate progenitor cells have the phenotype of high expression of CD49f protein, positive expression of Trop2 protein, low expression of CD26 protein and negative expression of CD117 protein.
  7. 7. The population of human prostate progenitor cells of claim 6, wherein high expression of the CD49f protein is defined as having a fluorescent signal intensity at the first 40% of the population of analyzed cells as measured by flow cytometry and low expression of the CD26 protein is defined as having a fluorescent signal intensity at the last 50% of the population of analyzed cells.
  8. 8. A pharmaceutical composition comprising the population of human prostate progenitor cells of claim 6 or 7 and a pharmaceutically acceptable lyoprotectant.
  9. 9. The pharmaceutical composition according to claim 8, wherein the lyoprotectant comprises 8% by weight/volume of trehalose, 5% by weight/volume of human serum albumin and 4% by weight/volume of polyvinylpyrrolidone K30, the composition is a lyophilized preparation, and the survival rate of the human prostate progenitor cells after resuscitation is not less than 85%.
  10. 10. Use of a population of human prostate progenitor cells according to claim 6 or 7 or a pharmaceutical composition according to any one of claims 8 or 9 for the preparation of a medicament for the treatment of chronic prostatitis, said medicament being administered by intra-prostate local injection.

Description

Stem cell preparation for treating prostate diseases and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to a stem cell preparation for treating a prostate disease, and a preparation method and application thereof. Background Benign prostatic hyperplasia, chronic prostatitis and tissue defect after treatment of prostate cancer are main causes of urination dysfunction and life quality degradation of middle-aged and elderly men. Current clinical treatments rely on α -receptor blockers or 5α -reductase inhibitors to alleviate lower urinary tract symptoms but fail to repair damaged glandular structures. Surgical procedures such as transurethral prostatectomy, while relieving obstruction, often result in partial loss of glands with long-term complications of urinary incontinence and sexual dysfunction. Therefore, the therapeutic means capable of realizing the structural regeneration and functional repair of the prostate tissue still remains a breakthrough in clinical urgent need. In recent years, regenerative medicine with stem cells as a core provides a new idea for prostate reconstruction. Among them, human induced pluripotent stem cells (hipscs) are considered as the starting cell source with the most promising clinical transformation prospect due to their unlimited expansion capacity and tricermal differentiation potential. Researchers have attempted to induce the differentiation of hipscs into prostate organoids or prostate epithelial-like cells by simulating embryonic development processes. The mainstream method generally follows the development path of 'definitive endoderm-hindgut-genitourinary sinus-prostate epithelium', and the sequential administration of Activin a, CHIR99021 induces the definitive endoderm, FGF4 and retinoic acid promote hindgut formation, and prostate specification is achieved by dihydrotestosterone, epidermal growth factor and fibroblast growth factor 10. Although this pathway demonstrates theoretical feasibility, three key challenges remain in clinical conversion. First, differentiation efficiency is low and product heterogeneity is high. The existing scheme mainly uses empirical growth factor combination, and lacks accurate space-time regulation and control on signal paths. For example, continued activation of BMP signaling inhibits the prostate lineage key factor NKX3-1, while the Wnt/β -catenin pathway, if of inappropriate intensity or window, would affect the expansion and specialization of prostate progenitor cells, resulting in lower target cell ratios and poor batch stability. Secondly, there is a lack of specific membrane surface markers that can be used for clinical grade non-destructive sorting and quality control. Currently, recognition of nuclear factors such as NKX3-1 and AR is mostly dependent, and therefore, the recognition of the nuclear factors is difficult to be used for sorting living cells. Although some of the prostate progenitor cells can be labeled by the reported CD49f and Trop2, the prostate tissue specificity is lacked, and the prostate tissue specificity is widely expressed in other epithelial stem cells, so that the purity and the safety cannot be ensured. Thirdly, the obtained cells have single functions and are difficult to cope with complex focus microenvironment. Areas of disease such as chronic prostatitis often involve high concentrations of inflammatory factors and immune cell infiltration, and "structural" progenitor cells obtained by traditional differentiation have low survival rates and easily inhibited functions in this environment. Meanwhile, the cells lack the immunoregulation capability, so that the local steady state reconstruction is difficult to promote, and the treatment effect is short. The intelligent therapeutic progenitor cells with the dual functions of immunoregulation and tissue regeneration become a breakthrough direction. In summary, there is a need for a systematic technical scheme starting from precise regulation of signal paths to realize efficient specialization, precise purification and functional optimization in prostate regeneration medicine. The establishment of the ready-to-use cell medicine which can be produced in a scale and standardized way is the key for realizing the structural regeneration and the functional recovery of the prostate tissue, and provides important technical support for improving the long-term curative effect and the life quality of male urinary system diseases. Disclosure of Invention First, the technical problem to be solved In the prior art, the preparation of the prostate therapeutic cells by utilizing the human pluripotent stem cells faces three main bottlenecks, namely, the differentiation scheme is low in efficiency and heterogeneous in products, high-purity specialization and sufficient amplification of a prostate lineage are difficult to realize simultaneously, secondly, the combinat